Compound Library and Method for Producing Compound Library

ABSTRACT

The present invention provides a method for producing a compound library comprising two or more cyclic compounds represented by the formula (I), comprising a step of allowing a macrocyclase in vitro to act on two or more peptides represented by the formula (II): LP-X—(Xa) m -Y—Z (II) wherein X represents a group represented by the formula (1), Y is a peptide residue consisting of four amino acids and/or analogs thereof and contains a group represented by the formula (2) (wherein R 1  and B 1  are as defined above, and R 3  represents a hydrogen or a hydrocarbon group), and LP is present or absent and, when present, represents a peptide residue consisting of 1 to 100 amino acids and/or analogs thereof, and forming the nitrogen-containing 6-membered ring A while eliminating LP, if present, to form the two or more cyclic compounds represented by the formula (I).

TECHNICAL FIELD

The present invention relates to a compound library and a method forproducing a compound library, etc.

BACKGROUND ART

With the development of genome sequencing technology in recent years, apeptide compound group called RiPPs (ribosomally synthesized andpost-translationally modified peptides) has received attention, which issynthesized in ribosomes and biosynthesized through post-translationalmodifications (Non-Patent Document 1). For RiPPs, biosynthetic genescalled structural gene which reside in biosynthetic gene clusters aretranslated by ribosomes to synthesize precursor peptides. Then, theprecursor peptides are subjected to a plurality of diverse modificationssuch as azole ring formation or dehydroamino acid formation bypost-translationally modifying enzymes encoded by other biosyntheticgenes, so that mature products are synthesized.

Thiopeptides, one of the RiPPs, have interesting physiological activitysuch as strong translation inhibitory activity, an antimicrobial effecton multi-drug-resistant organisms, an antiparasitic effect, an antiviraleffect, an immunosuppressive effect, an anticancer effect and the like.For examples, lactazoles A, B and C are known as the thiopeptides. Ithas also been reported that lazA-F, a lactazole biosynthetic genecluster, was found in the genome of an actinomycete Streptomyceslactacystinaeus (Non-Patent Document 2).

Thiopeptides have interesting activity, as mentioned above, andtherefore have received attention as medicament candidates or tools forresearch. Various attempts have been made to develop librariescontaining peptides having diverse structures and screen forthiopeptides having affinity for target substances.

An attempt to prepare thiopeptide analogs through the use of a mechanismof thiopeptide biosynthesis has been made by Walsh et al., who haveprepared thiopeptide analogs in vivo by introducing mutations tostructural genes using GE37468 and a thiocillin biosynthesis pathway(Non-Patent Documents 3 and 4).

In Non-Patent Document 4, Bacillus cereus ATCC 14579 is used as a hostfor thiocillin. In this document, an attempt has been made to prepareanalogs so as to change the size of the macrocycle. As Thiocillin has amacrocyclic structure of a 26-membered ring, it has been reported thatanalogs having a 23-, 26-, 29-, 32-, or 35-membered macrocycle were ableto be prepared by adding mutations to structural genes.

In vitro methods are known as methods for obtaining a library ofcompounds having a peptide structure.

Cell-free translation systems are systems for synthesizing peptides orproteins of interest in vitro through the use of protein-synthesisfunctions extracted from cells. The cell-free translation systemsinclude systems directly using translational factors extracted fromcells such as rabbit reticulocytes, wheat germs or E. coli, and systemscalled reconstituted cell-free translation systems, which prepare 31kinds of proteins for translation other than a ribosome one by one asrecombinant proteins and mix these proteins (Non-Patent Document 5).

The reconstituted cell-free translation systems, as compared with thesystems directly using cell extracts, have the advantage that peptidesor proteins can be synthesized with high efficiency because of involvingneither nuclease nor protease.

The cell-free translation systems are excellent in convenience becausetheir approaches for synthesizing proteins, as compared with chemicalsynthesis methods or production methods based on genetically recombinantorganisms, etc., have a rapid synthesis speed. Hence, the cell-freetranslation systems are also employed in drug discovery research. Forexample, “flexible in vitro translation system (FIT system)” is known(Non-Patent Document 6). The FIT system can construct a system forpreparing peptides containing non-proteinogenic amino acids by acell-free translation system, and a special peptide library having acyclic structure or the like by using this system.

CITATION LIST Non-Patent Document

-   Non-Patent Document 1: Arnison, P. G. et al. Ribosomally synthesized    and post-translationally modified peptide natural products: overview    and recommendations for a universal nomenclature. Nat Prod Rep 30,    108-160, (2013).-   Non-Patent Document 2: Hayashi, S. et al. Genome mining reveals a    minimum gene set for the biosynthesis of 32-membered macrocyclic    thiopeptides lactazoles. Chem Biol 21, 679-688, (2014).-   Non-Patent Document 3: Young, T. S., Dorrestein, P. C. &    Walsh, C. T. Codon randomization for rapid exploration of chemical    space in thiopeptide antibiotic variants. Chem Biol 19, 1600-1610,    (2012).-   Non-Patent Document 4: Bowers, A. A., Acker, M. G., Young, T. S. &    Walsh, C. T. Generation of thiocillin ring size variants by    prepeptide gene replacement and in vivo processing by Bacillus    cereus. J Am Chem Soc 134, 10313-10316, (2012).-   Non-Patent Document 5: Shimizu, Y. et al. Cell-free translation    reconstituted with purified components. Nat Biotechnol 19, 751-755,    (2001).-   Non-Patent Document 6: Goto, Y., Katoh, T. & Suga, H. Flexizymes for    genetic code reprogramming. Nat Protoc 6, 779-790, (2011).

SUMMARY Technical Problem

In cell-free translation systems, peptides or proteins of interest canbe synthesized in just 30 minutes, and, if many DNA or mRNA templatescan be synthesized, many types of peptides or proteins can be readilysynthesized according thereto. Thus, the cell-free translation systemsare useful for obtaining compound libraries because many types ofproteins can be synthesized conveniently and rapidly. However, anyapproach has not yet been known, which involves constructing a peptidecompound library having diverse structures in vitro by exploiting theadvantages of the cell-free translation systems and linking thecell-free translation systems to thiopeptide-biosynthesis systems.

Non-Patent Document 2 discloses that lazA-F, a lactazole biosyntheticgene cluster, was found. Nonetheless, an enzyme LazB-F expressed fromlazB-F has not been functionally determined. Besides, this document doesnot disclose in vitro reaction using these enzymes.

The possibility (substrate tolerance) is unclear thatthiopeptide-biosynthetic enzymes such as the enzyme LazB-F may formthiopeptides from diverse amino acid sequences including non-naturalsequences. Also, the possibility is unknown that thethiopeptide-biosynthetic enzymes can be applied to library construction.

The present invention has been made in light of the problems describedabove. An objective of the present invention is to provide a method forproducing a peptide compound library using a thiopeptide-biosynthesissystem.

Solution to Problem

The present inventors have conducted diligent studies to attain theobjective and consequently completed the present invention by findingthat: peptide compounds having diverse structures can be synthesized byusing predetermined thiopeptide-biosynthetic enzymes; and peptidecompound libraries can be developed using cell-free translation systemsand the thiopeptide-biosynthesis systems.

Specifically, the present invention is as follows.

[1]

A method for producing a compound library comprising two or more cycliccompounds represented by the formula (I):

wherein

m number of X_(a), and X_(b) and X_(c) each independently represent anamino acid residue selected from the group consisting of any amino acidsand analogs thereof,

Z is present or absent and, when present, represents a peptideconsisting of 1 to 100 amino acids and/or analogs thereof,

m is an integer selected from 2 to 40,

ring A is a nitrogen-containing 6-membered ring optionally having asubstituent,

B¹ is an oxygen atom, a sulfur atom, or a NH group, and

R¹ is a hydrogen atom or a hydrocarbon group,

comprising a step of allowing a macrocyclase in vitro to act on two ormore peptides represented by the formula (II):

LP-X—(Xa)_(m)-Y—Z  (II)

wherein

X represents a group represented by the formula (1):

(wherein R² is a hydrogen atom or a hydrocarbon group),

Y is a peptide residue consisting of four amino acids and/or analogsthereof and contains a group represented by the formula (2):

(wherein R¹ and B¹ are as defined above, and R³ represents a hydrogen ora hydrocarbon group),

m number of X_(a), m and Z are as defined above, and

LP is present or absent and, when present, represents a peptide residueconsisting of 1 to 100 amino acids and/or analogs thereof,

and forming the nitrogen-containing 6-membered ring A while eliminatingLP, if present, to form the two or more cyclic compounds represented bythe formula (I).

[2]

The production method according to [1], wherein LP is a peptide residueconsisting of 11 or more and 100 or less amino acids and/or analogsthereof.

[3]

The production method according to [1] or [2], wherein m is an integerselected from 2 to 24.

[4]

The production method according to any of [1] to [3], wherein Y is agroup represented by the following formula (3):

wherein R¹, R³, R⁴ and R⁵ each independently represent a hydrogen atomor a hydrocarbon group, and B¹ and B² each independently represent anoxygen atom, a sulfur atom, or a NH group.[5]

The production method according to any of [1] to [4], wherein Y is agroup represented by the following formula (3′):

wherein R¹, R³, R⁴ and R⁵ each independently represent a hydrogen atomor a methyl group, and B¹ and B² each independently represent an oxygenatom or a sulfur atom, with the proviso that when B¹ and/or B² is asulfur atom, each of R¹ and R⁵ is a hydrogen.[6]

The production method according to any of [1] to [5], wherein Y is agroup represented by the following formula (3″):

wherein R¹, R⁴ and R⁵ each independently represent a hydrogen atom or amethyl group, and B¹ and B² each independently represent an oxygen atomor a sulfur atom, with the proviso that when B¹ and/or B² is a sulfuratom, each of R¹ and R⁵ is a hydrogen.[7]

The production method according to any of [1] to [6], wherein Y is agroup represented by the following formula (3-1):

[8]

The production method according to any of [1] to [7], wherein themacrocyclase comprises LazC and/or an enzyme having homology with LazC.

[9]

A method for producing a compound library comprising two or more cycliccompounds represented by the formula (I):

wherein

m number of X_(a), and X_(b) and X_(c) each independently represent anamino acid residue selected from the group consisting of any amino acidsand analogs thereof,

Z is present or absent and, when present, represents a peptideconsisting of 1 to 100 amino acids and/or analogs thereof,

m is an integer selected from 2 to 40,

ring A is a nitrogen-containing 6-membered ring optionally having asubstituent,

B¹ is an oxygen atom, a sulfur atom, or a NH group, and

R¹ is a hydrogen atom or a hydrocarbon group, the method comprising:

a step of producing a mRNA library encoding precursor peptidesrepresented by the formula (III):

$\begin{matrix}{{LP}^{\prime} - X^{\prime} - ( {Xa}^{\prime} )_{m^{\prime}} - Y^{\prime} - Z^{\prime}} & ({III})\end{matrix}$

wherein

X′ is serine or threonine, or an analog thereof,

Y′ is a peptide consisting of four amino acids represented by—Y′(10)-Y′(11)-Y′(12)-Y′(13)- and/or analogs thereof, wherein Y′(10) isan amino acid residue selected from the group consisting of any aminoacids and analogs thereof, Y′(11) is serine, cysteine, threonine, ordiaminopropionic acid or an analog thereof, Y′(12) is serine orthreonine or an analog thereof, and Y′(13) is an amino acid residueselected from the group consisting of any amino acids and analogsthereof,

m′ number of Xa′, m′ and Z′ are the same as defined as m number of Xa, mand Z, respectively, in the formula (I), and

LP′ is present or absent and, when present, represents a peptideconsisting of 1 to 100 amino acids and/or analogs thereof;

a step of expressing the precursor peptides by a cell-free translationsystem with the mRNA library to produce a first peptide library;

a step of reacting an enzyme for forming azole ring(s) with the firstpeptide library, so that at least an azole ring is formed on Y′(11) inthe precursor peptides to produce a second peptide library;

a step of reacting an enzyme for forming α,β-unsaturated amino acid(s)with the second peptide library in the presence of cosubstratetRNA^(Glu) for glutamylation reaction and aminoacylation enzyme GluRS,so that at least X′ and Y′(12) in the precursor peptides are convertedto α,β-unsaturated amino acid residues to produce a third peptidelibrary; and

a step of reacting a macrocyclase with the third peptide library andforming a nitrogen-containing 6-membered ring while eliminating LP′, ifpresent, to form the two or more cyclic compounds represented by theformula (I).

[10]

A method for producing a compound library comprising two or more cycliccompounds represented by the formula (I):

wherein

m number of X_(a), and X_(b) and X_(c) each independently represent anamino acid residue selected from the group consisting of any amino acidsand analogs thereof,

Z is present or absent and, when present, represents a peptideconsisting of 1 to 100 amino acids and/or analogs thereof,

m is an integer selected from 2 to 40,

ring A is a nitrogen-containing 6-membered ring optionally having asubstituent,

B¹ is an oxygen atom, a sulfur atom, or a NH group, and

R¹ is a hydrogen atom or a hydrocarbon group, the method comprising:

a step of producing a mRNA library encoding precursor peptidesrepresented by the formula (III):

$\begin{matrix}{{LP}^{\prime} - X^{\prime} - ( {Xa}^{\prime} )_{m^{\prime}} - Y^{\prime} - Z^{\prime}} & ({III})\end{matrix}$

wherein

X′ is serine or threonine, or an analog thereof,

Y′ is a peptide consisting of four amino acids represented by—Y′(10)-Y′(11)-Y′(12)-Y′(13)- and/or analogs thereof, wherein Y′(10) isan amino acid residue selected from the group consisting of any aminoacids and analogs thereof, Y′(11) is serine, cysteine, threonine, ordiaminopropionic acid or an analog thereof, Y′(12) is serine orthreonine or an analog thereof, and Y′(13) is an amino acid residueselected from the group consisting of any amino acids and analogsthereof,

m′ number of Xa′, m′ and Z′ are as the same defined as m number of Xa, mand Z, respectively, in the formula (I), and

LP′ is present or absent and, when present, represents a peptideconsisting of 1 to 100 amino acids and/or analogs thereof;

a step of binding puromycin to the 3′ end of each mRNA of the mRNAlibrary to produce a puromycin-bound mRNA library;

a step of expressing the precursor peptides by a cell-free translationsystem with the puromycin-bound mRNA library to produce a firstpeptide-mRNA complex library;

a step of reacting an enzyme for forming azole ring(s) with the firstpeptide-mRNA complex library, so that an azole ring is formed on atleast Y′(11) in the precursor peptides to produce a second peptide-mRNAcomplex library;

a step of reacting an enzyme for forming α,β-unsaturated amino acid(s)with the second peptide-mRNA complex library in the presence ofcosubstrate tRNA^(Glu) for glutamylation reaction and aminoacylationenzyme GluRS, so that at least X′ and Y′(12) in the precursor peptidesare converted to α,β-unsaturated amino acid residues to produce a thirdpeptide-mRNA complex library; and

a step of reacting a macrocyclase with the third peptide-mRNA complexlibrary and forming a nitrogen-containing 6-membered ring whileeliminating LP′, if present, to form the two or more cyclic compoundsrepresented by the formula (I).

[11]

The production method according to [9] or [10], wherein LP′ is a peptideconsisting of 11 or more and 100 or less amino acids and/or analogsthereof.

[12]

The production method according to any of [9] to [11], wherein m is aninteger selected from 2 to 24.

[13]

The production method according to any of [9] to [12], wherein

Y′(10) is serine or threonine or an analog thereof, and

Y′(13) is serine, cysteine, threonine, or diaminopropionic acid or ananalog thereof.

[14]

The production method according to any of [9] to [13], wherein

Y′(11) is serine, cysteine, or threonine, or an analog thereof, and

Y′(12) is serine or threonine, or an analog thereof.

[15]

The production method according to any of [9] to [14], wherein

Y′(11) is serine, cysteine, or threonine, or an analog thereof, and

Y′(12) is serine or an analog thereof.

[16]

The production method according to any of [9] to [15], wherein

Y′(10) is serine or threonine or an analog thereof,

Y′(11) is serine, cysteine, or threonine, or an analog thereof,

Y′(12) is serine, and

Y′(13) is serine, cysteine, or threonine, or an analog thereof.

[17]

The production method according to any of [9] to [16], wherein in(Xa′)_(m), in the formula (III), amino acid residue Xa′(1) adjacent toX′ is an amino acid other than an acidic amino acid or an analogthereof.

[18]

The production method according to any of [9] to [17], wherein in(Xa′)_(m), in the formula (III), amino acid residue Xa′(m′) at m′-thposition is an amino acid other than an acidic amino acid or an analogthereof.

[19]

The production method according to any of [9] to [18], wherein in(Xa′)_(m), in the formula (III), amino acid residue Xa′(m′−1) at(m′−1)-th position is an amino acid other than an acidic amino acid anda basic amino acid or an analog thereof.

[20]

The production method according to any of [9] to [19], wherein theenzyme for forming azole ring(s) comprises LazD, LazE and LazF, and/orenzymes having homology with any of them.

[21]

The production method according to any of [9] to [20], wherein theenzyme for forming α,β-unsaturated amino acid(s) comprises LazB andLazF, and/or enzymes having homology with any of them.

[22]

The production method according to any of [9] to [21], wherein themacrocyclase comprises LazC and/or an enzyme having homology with LazC.

[23]

The production method according to any of [9] to [22], wherein

the cosubstrate tRNA^(Glu) for glutamylation reaction isactinomycete-derived tRNA^(Glu), and

the aminoacylation enzyme GluRS is actinomycete-derived GluRS.

[24]

A compound library comprising two or more cyclic compounds representedby the formula (I):

wherein

m number of X_(a), and X_(b) and X_(c) each independently represent anamino acid residue selected from the group consisting of any amino acidsand analogs thereof,

Z is present or absent and, when present, represents a peptideconsisting of 1 to 100 amino acids and/or analogs thereof,

m is an integer selected from 2 to 40,

ring A is a nitrogen-containing 6-membered ring optionally having asubstituent,

B¹ is an oxygen atom, a sulfur atom, or a NH group, and

R¹ is a hydrogen atom or a hydrocarbon group, wherein

at least one of the cyclic compounds represented by the formula (I) isin a non-natural form.

[25]

A screening method for identifying a cyclic compound represented by theformula (I) binding to a target substance, comprising:

a step of contacting a compound library produced by the productionmethod according to any of [1] to [23] or the compound library accordingto [24] with the target substance, followed by incubation; and

a step of selecting a cyclic compound represented by the formula (I)bound to the target substance.

[26]

A screening kit for identifying a cyclic compound represented by theformula (I) binding to a target substance,

comprising a compound library produced by the production methodaccording to any of [1] to [23] or the compound library according to[24].

[27]

A method for producing a cyclic compound represented by the formula (I):

wherein

m number of X_(a), and X_(b) and X_(c) each independently represent anamino acid residue selected from the group consisting of any amino acidsand analogs thereof,

Z is present or absent and, when present, represents a peptideconsisting of 1 to 100 amino acids and/or analogs thereof,

m is an integer selected from 2 to 40,

ring A is a nitrogen-containing 6-membered ring optionally having asubstituent,

B¹ is an oxygen atom, a sulfur atom, or a NH group, and

R¹ is a hydrogen atom or a hydrocarbon group, comprising a step ofallowing a macrocyclase in vitro to act on a peptide represented by theformula (II):

$\begin{matrix}{{LP} - X - ( {Xa} )_{m} - Y - Z} & ({II})\end{matrix}$

wherein

X represents a group represented by the formula (1):

(wherein R² is a hydrogen or a hydrocarbon group),

Y is a peptide consisting of four amino acids and/or analogs thereof andcontains a group represented by the formula (2):

(wherein R¹ and R³ each represent hydrogen or a hydrocarbon group, andB¹ represents an oxygen atom, a sulfur atom, or a NH group),

m number of X_(a), m and Z are as defined above, and

LP is present or absent and, when present, represents a peptide residueconsisting of 1 to 100 amino acids and/or analogs thereof,

and forming the nitrogen-containing 6-membered ring A while eliminatingLP, if present, to form the cyclic compound represented by the formula(I).

[28]

A method for producing a cyclic compound represented by the formula (I):

wherein

m number of X_(a), X_(b), and X_(c) each independently represent anamino acid residue selected from the group consisting of any amino acidsand analogs thereof,

Z is present or absent and, when present, represents a peptideconsisting of 1 to 100 amino acids and/or analogs thereof,

m is an integer selected from 2 to 40,

ring A is a nitrogen-containing 6-membered ring optionally having asubstituent,

B¹ is an oxygen atom, a sulfur atom, or a NH group, and

R¹ is a hydrogen atom or a hydrocarbon group, the method comprising:

a step of producing mRNA encoding a precursor peptide represented by theformula (III):

$\begin{matrix}{{LP}^{\prime} - X^{\prime} - ( {Xa}^{\prime} )_{m^{\prime}} - Y^{\prime} - Z^{\prime}} & ({III})\end{matrix}$

wherein

X′ is serine or threonine, or an analog thereof,

Y′ is a peptide consisting of four amino acids represented by—Y′(10)-Y′(11)-Y′(12)-Y′(13)- and/or analogs thereof, wherein Y′(10) isan amino acid residue selected from the group consisting of any aminoacids and analogs thereof, Y′(11) is serine, cysteine, threonine, ordiaminopropionic acid or an analog thereof, Y′(12) is serine orthreonine or an analog thereof, and Y′(13) is an amino acid residueselected from the group consisting of any amino acids and analogsthereof,

m′ number of Xa′, m′ and Z′ are the same as defined as m number of Xa, mand Z, respectively, in the formula (I), and

LP′ is present or absent and, when present, represents a peptideconsisting of 1 to 100 amino acids and/or analogs thereof;

a step of expressing the precursor peptide by a cell-free translationsystem with the mRNA to produce a first peptide;

a step of reacting an enzyme for forming azole ring(s) with the firstpeptide, so that an azole ring is formed on at least Y′(11) in theprecursor peptide to produce a second peptide;

a step of reacting an enzyme for forming α,β-unsaturated amino acid(s)with the second peptide in the presence of cosubstrate tRNA^(Glu) forglutamylation reaction and aminoacylation enzyme GluRS, so that at leastX′ and Y′(12) in the precursor peptide are converted to α,β-unsaturatedamino acid residues to produce a third peptide; and

a step of reacting a macrocyclase with the third peptide and forming anitrogen-containing 6-membered ring while eliminating LP′, if present,to form a cyclic compound represented by the formula (I).

According to the present invention, a compound library much more diversethan that prepared by synthesis or in vivo can be obtained by a rapidand convenient method. Use of this compound library enables a compoundbinding to a target substance to be screened for.

Furthermore, a mRNA display method can be applied to the compoundlibrary of the present invention to prepare a library of complexes withmRNAs encoding the peptide moieties of the compounds, so that nucleicacid sequences encoding compounds identified by screening can bedetermined, and the relationship between the structures and activitiesof the compounds can be readily analyzed.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a diagram showing the sequences of LazA and LazA*.

FIG. 2 is a schematic view of a lazA DNA template. This templatecontains a T7 promoter sequence, a translation enhancer sequence, a SDsequence and a DNA template encoding LazA* and has a total length of 214base pairs.

FIG. 3 is a diagram showing the nucleotide sequence of lazA havingcodons changed to E. coli type.

FIG. 4 is a schematic view of primer extension and PCR.

FIG. 5 is a diagram showing the alignment of tRNA^(Glu) nucleotidesequences of E. coli and lactazole-producing microbes. The 72nd and 73rdbases, which were important for the substrate recognition of MibB, areemphasized with a box.

FIG. 6 is a diagram showing a tRNA^(Glu) DNA template (a total length of97 bp) for in vitro transcription.

FIG. 7 is a diagram showing results of performing azole ring formationreaction (5 hr) followed by α,β-unsaturated amino acid formationreaction. A. A reaction system of LazA* alone; B. A system in whichLazD, LazE and LazF were added to LazA* and reacted for 5 hours; and C.A reaction system in which LazB, LazF, tRNA^(Glu) (CUC) and GluRS wereadded after the reaction of B. It is considered that: m/z 5656.5 isderived from a peptide obtained by the Michael addition of DTT(molecular weight: 154) to dehydroalanine of m/z 5502.5; and m/z 5674.5is derived from a peptide obtained by the Michael addition of DTT(molecular weight: 154) to a tetradehydrate.

FIG. 8 is a diagram showing results of performing α,β-unsaturated aminoacid formation reaction (5 hr) followed by azole ring formationreaction. A. A reaction system of LazA* alone; B. A system in whichLazB, LazF, tRNA^(Glu) (CUC) and GluRS were added to LazA* and reactedfor 5 hours; and C. A reaction system in which LazD, LazE and LazF wereadded after the reaction of B.

FIG. 9 is a diagram showing results of reaction of LazC (m/z 2000 to).A. A reaction system of LazA* alone; B. A reaction system in which LazD,LazE and LazF were added to LazA*; C. A reaction system in which LazD,LazE, LazF, LazB, tRNA^(Glu) and GluRS were added to LazA*; and D. Areaction system in which LazD, LazE, LazF, LazB, tRNA^(Glu), GluRS andLazC were added to LazA*.

FIG. 10 shows that the same MS spectra as those of FIG. 9D in the rangeof m/z 1415 to 1435 were enlarged and overlaid. A signal of a Na adduct(calcd. 1423.369) of lactazole A was specifically seen in the system ofLazC(+).

FIG. 11 shows the LC-MS chromatograms (EIC) of leader peptides cleavedoff during Ala-inserted analog formation.

FIG. 12 is a diagram showing the peak areas of leader peptides cleavedoff during Ala-inserted analog formation.

FIG. 13 shows the LC-MS chromatograms (EIC) of leader peptides cleavedoff during Ala multiply-inserted analog and -deleted analog formation.

FIG. 14 is a diagram showing the peak areas of leader peptides cleavedoff during Ala multiply-inserted analog and -deleted analog formation.

FIG. 15 shows the LC-MS chromatogram (EIC) of a leader peptide cleavedoff during 4SCSC7_4AAAA7 analog formation.

FIG. 16 shows the LC-MS chromatograms (EIC) of leader peptides cleavedoff through LazC reaction with Ala-substituted precursor peptides assubstrates.

FIG. 17 shows the LC-MS chromatograms (EIC) of reaction products by Lazenzyme group with a Trp(2) or Gly(3)-substituted analog as a substrate.

FIG. 18 shows the LC-MS chromatograms (EIC) of reaction products by Lazenzyme group with a Gln(8) or Ala(9)-substituted analog as a substrate.

FIG. 19 shows the LC-MS chromatograms (EIC) of reaction products by Lazenzyme group with a Gln(15) or Pro(16)-substituted analog as asubstrate.

FIG. 20 is a diagram showing results of reaction in substituting aresidue (Trp(2), Gly(3), Gln(8), Ala(9), Gln(15) or Pro(16)) in amacrocycle.

FIG. 21 shows the LC-MS chromatograms (EIC) of reaction products by Lazenzyme group with Ala multiply-inserted precursor peptides assubstrates.

FIG. 22 shows the LC-MS chromatograms (EIC) of reaction products by Lazenzyme group with multiply-deleted precursor peptides as a substrate.

FIG. 23 shows the peak areas of leader peptides cleaved off fromreaction products by Laz enzyme group with Ala multiply-insertedprecursor peptides as substrates.

FIG. 24 shows the peak areas of leader peptides cleaved off fromreaction products by Laz enzyme group with multiply-deleted precursorpeptides as substrates.

FIG. 25 shows the LC-MS chromatograms (brEIC) of reaction productsmediated by a Laz enzyme group with Ala and Asn multiply-insertedprecursor peptides or multiply-deleted precursor peptides as substrates.

FIG. 26 shows the LC-MS chromatograms (brEIC) of reaction productsmediated by a Laz enzyme group for Ala-substituted analogs.

FIG. 27 shows the LC-MS chromatograms (brEIC) of reaction productsmediated by a Laz enzyme group for Lys-substituted analogs.

FIG. 28 shows the LC-MS chromatograms (brEIC) of reaction productsmediated by a Laz enzyme group for Glu-substituted analogs.

FIG. 29 is a diagram showing the LC-MS chromatograms (EIC) of leaderpeptides cleaved off through LazC reaction with a Ser(10), Ser(11) orCys(13)-substituted analog as a substrate.

FIG. 30 shows the LC-MS chromatograms (brEIC) of reaction productsmediated by a Laz enzyme group for analogs (substitution and extension)as to a C-terminal region.

FIG. 31 shows the LC-MS chromatograms (brEIC) of reaction productsmediated by a Laz enzyme group for analogs having a randomizedintra-ring sequence, and an analog having a randomized intra-ringsequence and an extended C-terminal region.

FIG. 32 shows results of azole ring formation reaction mediated by LazD,LazE and LazF, and α,β-unsaturated amino acid formation reactionmediated by LazB and LazF when LazA* was used as a substrate.

FIG. 33 shows results of azole ring formation reaction mediated by LazD,LazE and LazF, and α,β-unsaturated amino acid formation reactionmediated by LazB and LazF when LP-30 was used as a substrate.

FIG. 34 shows results of azole ring formation reaction mediated by LazD,LazE and LazF, and α,β-unsaturated amino acid formation reactionmediated by LazB and LazF when LP-25 was used as a substrate.

FIG. 35 shows results of azole ring formation reaction mediated by LazD,LazE and LazF, and α,β-unsaturated amino acid formation reactionmediated by LazB and LazF when LP-20 was used as a substrate.

FIG. 36 shows results of azole ring formation reaction mediated by LazD,LazE and LazF, and α,β-unsaturated amino acid formation reactionmediated by LazB and LazF when LP-15 was used as a substrate.

FIG. 37 shows the LC-MS chromatograms (EIC) of leader peptides usingleader peptide-truncated precursor peptides as substrates.

FIG. 38 is a schematic view of altered precursor peptides prepared bysubstituting each of their amino acid residues positioned from −25 to −1of a leader peptide in LP-25 by Ala.

FIG. 39 is a diagram showing the LC-MS chromatograms (EIC) of leaderpeptides in reaction products of altered precursor peptides prepared bythe substitution of each of their amino acid residues positioned from−25 to −1.

FIG. 40 is a diagram showing the design of compound libraries.

FIG. 41 is a diagram of an electrophoretic gel showing that mRNAlibraries were prepared. In the diagram, 6 to 11 denote mRNA librariescontaining any amino acid sequences consisting of 5, 6, 7, 8, 9 and 10residues, respectively.

FIG. 42 is a graph showing sequencing quality score Q in sequencing theDNA sequences of reverse transcription products of prepared mRNAlibraries.

FIG. 43 is a diagram showing results of evaluating the lengths ofprepared mRNA libraries and analyzing the degree of frameshift.

FIG. 44 is a diagram showing results of evaluating the sequenceconservation of prepared mRNA libraries in order to confirm that:invariable regions of the libraries correctly had particular sequences;and variable regions evenly had random sequences.

FIG. 45 is a diagram showing results of analyzing the frequency of eachamino acid in random regions contained in prepared mRNA libraries.

FIG. 46 is a diagram showing a scheme in obtaining a cyclic peptidebinding to iPGM, comprising a step of constructing a peptide-mRNAcomplex library from a mRNA library and further constructing a compoundlibrary containing cyclic peptides by the action of LazB-F enzyme.

FIG. 47 is a diagram showing a graph of the rate of recovery of cDNAobtained by a selection experiment carried out by the scheme of FIG. 52.

FIG. 48 is a schematic view showing four types of codons reprogrammablefor the introduction of non-proteinogenic amino acid(s) in thepreparation of analogs containing the non-proteinogenic amino acid(s).

FIG. 49 is a schematic view of DNA templates provided for thepreparation of analogs containing non-proteinogenic amino acid(s). tggand aag codons corresponding to the boxed W and K were reprogrammed tointroduce non-proteinogenic amino acid(s) to a substrate peptide.

FIG. 50 shows the LC-MS chromatograms (brEIC) of reaction productsmediated by a Laz enzyme group for non-proteinogenic aminoacid-substituted analog N^(Me)Gly.

FIG. 51 shows the LC-MS chromatograms (brEIC) of reaction productsmediated by a Laz enzyme group for non-proteinogenic aminoacid-substituted analog N^(Me)Ala.

FIG. 52 is a schematic view showing a experimental procedure forrevealing that a precursor peptides displayed on mRNA is modified by alactazole biosynthetic enzyme.

FIG. 53 is a diagram showing the LC-MS chromatogram of a leader peptidecleaved off through LazC reaction with a peptide-mRNA complex as asubstrate.

FIG. 54 is a diagram showing results of reaction in enzyme additionpatterns I, II, III and IV.

DESCRIPTION OF EMBODIMENTS

Hereinafter, embodiments of the present invention will be described indetail. However, the present invention is not limited by the followingpresent embodiment and can be carried out through various changes ormodifications without departing from the spirit of the presentinvention.

One embodiment of the method for producing a compound library accordingto the present invention is a method for producing a compound librarycontaining two or more cyclic compounds represented by the formula (I):

wherein

m number of X_(a), and X_(b) and X_(c) each independently represent anamino acid residue selected from the group consisting of any amino acidsand analogs thereof,

Z is present or absent and, when present, represents a peptideconsisting of 1 to 100 amino acids and/or analogs thereof,

m is an integer selected from 2 to 40,

ring A is a nitrogen-containing 6-membered ring optionally having asubstituent,

B¹ is an oxygen atom, a sulfur atom, or a NH group, and

R¹ is a hydrogen atom or a hydrocarbon group, the method including astep of allowing a macrocyclase in vitro to act on two or more peptidesrepresented by the formula (II):

$\begin{matrix}{{LP} - X - ( {Xa} )_{m} - Y - Z} & ({II})\end{matrix}$

wherein

X represents a group represented by the formula (1):

(wherein R² is a hydrogen atom or a hydrocarbon group),

Y is a peptide residue consisting of four amino acids and/or analogsthereof and contains a group represented by the formula (2):

(wherein R¹ and R³ each represent a hydrogen or a hydrocarbon group, andB¹ represents an oxygen atom, a sulfur atom, or a NH group),

m number of X_(a) each independently represents an amino acid residueselected from the group consisting of any amino acids and analogsthereof,

LP is present or absent and, when present, represents a peptideconsisting of 1 to 100 amino acids and/or analogs thereof,

Z is present or absent and, when present, represents a peptideconsisting of 1 to 100 amino acids and/or analogs thereof, and

m is an integer selected from 2 to 40,

and forming the nitrogen-containing 6-membered ring A while eliminatingLP, if present, to form the two or more cyclic compounds represented bythe formula (I).

In the formula (I) and the formula (II), m number of X_(a) may be thesame or different and each independently represents an amino acidresidue selected from the group consisting of any amino acids andanalogs thereof.

In the formula (1) and the formula (2), the unsaturated double bondcontaining R² or R³ may be a single isomer in E or Z form or may be amixture of E and Z forms. The formula (1) is preferably represented by

and the formula (2) is preferably represented by

One embodiment of the method for producing a compound library accordingto the present invention is a method for producing a compound librarycontaining two or more cyclic compounds represented by the formula (I),including:

a step of producing a mRNA library encoding precursor peptidesrepresented by the formula (III):

$\begin{matrix}{{LP}^{\prime} - X^{\prime} - ( {Xa}^{\prime} )_{m^{\prime}} - Y^{\prime} - Z^{\prime}} & ({III})\end{matrix}$

wherein

X′ is serine or threonine, or an analog thereof,

Y′ is a peptide consisting of four amino acids represented by—Y′(10)-Y′(11)-Y′(12)-Y′(13)- and/or analogs thereof, wherein Y′(10) isan amino acid residue selected from the group consisting of any aminoacids and analogs thereof, Y′(11) is serine, cysteine, threonine, ordiaminopropionic acid or an analog thereof, Y′(12) is serine orthreonine or an analog thereof, and Y′(13) is an amino acid residueselected from the group consisting of any amino acids and analogsthereof,

m′ number of X_(a)′ each independently represents an amino acid residueselected from the group consisting of any amino acids and analogsthereof,

LP′ is present or absent and, when present, represents a peptideconsisting of 1 to 100 amino acids and/or analogs thereof,

Z′ is present or absent and, when present, represents a peptideconsisting of 1 to 100 amino acids and/or analogs thereof, and

m′ is an integer selected from 2 to 40;

a step of expressing the precursor peptides by a cell-free translationsystem with the mRNA library to produce a first peptide library;

a step of reacting an azole ring-forming enzyme with the first peptidelibrary, so that an azole ring is formed on at least Y′(11) in theprecursor peptides to produce a second peptide library; and

a step of reacting an α,β-unsaturated amino acid-forming enzyme with thesecond peptide library in the presence of cosubstrate tRNA^(Glu) forglutamylation reaction and aminoacylation enzyme GluRS, so that at leastX′ and Y′(12) in the precursor peptides are converted to α,β-unsaturatedamino acid residues to produce a third peptide library; and

a step of reacting a macrocyclase with the third peptide library andforming a nitrogen-containing 6-membered ring while eliminating LP′, ifpresent, to form the two or more cyclic compounds represented by theformula (I).

In the formula (III), m′ number of X_(a)′ may be the same or differentand each independently represents an amino acid residue selected fromthe group consisting of any amino acids and analogs thereof.

The “α,β-unsaturated amino acid” in the α,β-unsaturated aminoacid-forming enzyme refers to an amino acid having an unsaturated doublebond formed between a carbon at position α and a carbon at position βadjacent to the carbon at position α in an amino acid or an analogthereof, as shown in [Formula 21] given below. The unsaturated doublebond may be a single isomer in E or Z form or may be a mixture of E andZ forms.

In the formula, R^(a) is any organic group.

One embodiment of the method for producing a compound library accordingto the present invention is a method for producing a compound librarycontaining two or more cyclic compounds represented by the formula (I),including:

a step of producing a mRNA library encoding precursor peptidesrepresented by the formula (III);

a step of binding puromycin to the 3′ end of each mRNA of the mRNAlibrary to produce a puromycin-bound mRNA library;

a step of expressing the precursor peptides by a cell-free translationsystem with the puromycin-bound mRNA library to produce a firstpeptide-mRNA complex library;

a step of reacting an azole ring-forming enzyme with the firstpeptide-mRNA complex library, so that an azole ring is formed on atleast Y′(11) in the precursor peptides to produce a second peptide-mRNAcomplex library;

a step of reacting an α,β-unsaturated amino acid-forming enzyme with thesecond peptide-mRNA complex library in the presence of cosubstratetRNA^(Glu) for glutamylation reaction and aminoacylation enzyme GluRS,so that at least X′ and Y′(12) in the precursor peptides are convertedto α,β-unsaturated amino acid residues to produce a third peptide-mRNAcomplex library; and

a step of reacting a macrocyclase with the third peptide-mRNA complexlibrary and forming a nitrogen-containing 6-membered ring whileeliminating LP′, if present, to form the two or more cyclic compoundsrepresented by the formula (I).

The method for producing a compound library according to the presentinvention can conveniently produce a compound library ofpost-translationally modified and macrocyclized compounds withoutincluding a step of removing leader peptides, whereas the leaderpeptides usually need to be removed for in vitro use of enzymes in apost-translational modification reaction step.

In the present specification, the “amino acid” is a proteinogenic aminoacid. In the present specification, the “proteinogenic amino acid” meansan amino acid constituting a protein (Arg, His, Lys, Asp, Glu, Ser, Thr,Asn, Gln, Cys, Gly, Pro, Ala, Ile, Leu, Met, Phe, Trp, Tyr, and Val). Inthe present specification, the amino acid is also indicated by aconventional single-letter code or three-letter code.

In the present specification, the “analog of an amino acid” includes anatural or non-natural amino acid other than a proteinogenic amino acid.The natural or non-natural amino acid other than a proteinogenic aminoacid is also referred to as a “non-proteinogenic amino acid”. Examplesof the non-proteinogenic amino acid include, but are not limited to:α,α-disubstituted amino acids (α-methylalanine, etc.), N-alkyl-α-aminoacids, D-amino acids, β-amino acids and α-hydroxylic acid, which differin backbone structure from natural forms; amino acids differing in sidechain structure from natural forms (norleucine, homohistidine, etc.);amino acids having extra methylene on a side chain (“homo” amino acids,homophenylalanine, diaminopropionic acid, β-methylcysteine,β-isopropylserine, β-phenylserine, etc.); and amino acids obtained bysubstituting a carboxylic acid functional group amino acid in a sidechain by sulfonic acid (cysteic acid, etc.). In the presentspecification, the “analog of an amino acid” also encompasses a mutantor a derivative of a proteinogenic amino acid or a non-proteinogenicamino acid and, specifically, also includes derivatives, such as anα,β-unsaturated amino acid and azole, formed from a proteinogenic aminoacid or a non-proteinogenic amino acid.

When the peptide according to the present invention contains anα,β-unsaturated amino acid as an amino acid analog, examples of theα,β-unsaturated amino acid as the amino acid analog specifically includeanalog (I) having the following structure:

wherein R^(X1) is a hydrogen atom or a hydrocarbon group.

When the peptide according to the present invention contains azole as anamino acid analog, examples of the azole as the amino acid analogspecifically include analog (II) having the following structure:

wherein B^(X) is an oxygen atom, a sulfur atom or a NH group, and R^(X2)is a hydrogen atom or a hydrocarbon group.

In the present specification, a chemical structural formula encompassesa tautomer, a geometric isomer, an optical isomer and the like.

Particularly, examples of the analog of Cys include, but are not limitedto, an analog represented by a formula given below. Alternatively, theanalog of Cys may be analog (I) or analog (II), as mentioned above,derived from the following formula:

wherein R represents a hydrogen atom, an alkyl group having 1 to 10carbon atoms and optionally having a substituent, or an aromatic groupoptionally having a substituent.

Particularly, examples of the analog of Ser or Thr include, but are notlimited to, an analog represented by a formula given below.Alternatively, the analog of Ser or Thr may be analog (I) or analog(II), as mentioned above, derived from the following formula:

wherein R represents a hydrogen atom, an alkyl group having 1 to 10carbon atoms and optionally having a substituent, or an aromatic groupoptionally having a substituent.

Particularly, examples of the analog of diaminopropionic acid (Dap)include, but are not limited to, an analog given below. Alternatively,the analog of diaminopropionic acid may be analog (I) or analog (II), asmentioned above, derived from the following formula:

wherein R represents a hydrogen atom, an alkyl group having 1 to 10carbon atoms and optionally having a substituent, or an aromatic groupoptionally having a substituent.

In the present specification, examples of the hydrocarbon grouppreferably include an alkyl group having 1 to 10 carbon atoms andoptionally having a substituent, and an aromatic group optionally havinga substituent. In the present specification, the hydrocarbon group ispreferably an alkyl group having 1 to 6 carbon atoms or a phenyl groupoptionally having a substituent, more preferably an alkyl group having 1to 4 carbon atoms or a phenyl group, and further preferably a methylgroup.

In the present specification, examples of the substituent include analkyl group, an aromatic group, a halogen group, an azide group and anamino group. The substituent is preferably an alkyl group having 1 to 4carbon atoms, and more preferably a methyl group.

The nitrogen-containing 6-membered ring optionally having a substituentin the formula (I) is not particularly limited as long as thenitrogen-containing 6-membered ring results from a reaction between theformula (1) and the formula (2), i.e., 2+4 cycloaddition. Specifically,examples thereof include the following structures:

wherein R^(A1) and R^(A2) are each independently hydrogen or ahydrocarbon group, and each of R^(A1) and R^(A2) is preferably ahydrogen or an alkyl group having 1 to 4 carbon atoms, and morepreferably a hydrogen or a methyl group.

Each of LP and LP′ is also referred to as a leader peptide andrepresents a peptide consisting of 1 to 100 amino acids and/or analogsthereof. LP and LP′ may be present or absent. The presence of LP or LP′tends to facilitate recognizing a precursor peptide by an enzyme such asa macrocyclase, an azole ring-forming enzyme, an α,β-unsaturated aminoacid-forming enzyme and the like, so that the reaction proceedssmoothly.

The number of amino acids and/or analogs thereof constituting LP or LP′is preferably 11 or more and 100 or less, more preferably 11 or more and60 or less, further preferably 16 or more and 45 or less, as to LP, andpreferably 11 or more and 100 or less, more preferably 11 or more and 60or less, further preferably 16 or more and 45 or less, as to LP′ forsufficient substrate recognition by a modifying enzyme.

An important leader peptide region for the azole ring-forming enzyme hasbeen revealed in goadsporin, which has a thiopeptide-like modifiedstructure (Ozaki, T. et al. Dissection of goadsporin biosynthesis by invitro reconstitution leading to designer analogues expressed in vivo.Nat Commun 8, 14207, doi:10.1038/ncomms14207 (2017)). Table 1 showsresults of a leader peptide truncation experiment for biosynthesis ofgoadsporin.

TABLE 1 Amino-acid sequence of LPs Entity Name

1 GodA*

2 GodA*LP

3 GodA*LP

4 GodA*LP

5 GodA*LP

6 GodA*LP

Number Number Amino-acid sequence of LPs of of Entity Name

azoles Dhas 1 GodA*

6 1, 2 2 GodA*LP

6 1, 2 3 GodA*LP

6 1, 2 4 GodA*LP

4, 5, 6 1 5 GodA*LP

0 0 6 GodA*LP

0 0

indicates data missing or illegible when filed

As shown in the table, leader-truncated precursor peptides obtained byN-terminally truncating a leader peptide region of precursor peptideGodA of goadsporin are used as substrates. For the leader peptide regionhaving 20 residues (LP-20) or more, six azole rings are formed. For theleader peptide region having 15 residues (LP-15), the efficiency ofazole ring formation is reduced. Azole ring formation is no longer seenin shorter ones (LP-10 and LP-5).

LP is not particularly limited by its sequence and is preferably apeptide of -(−20th)LSELTVTSLRDTVALPENGA(−1st)- from the −20th to −1st(the −1st amino acid is adjacent to X) amino acids in order from the Nterminus with X in the formula (II) defined as position 1, or a peptidehaving homology with this peptide. For LP, the homology with the peptideof -LSELTVTSLRDTVALPENGA- is preferably 50% or more, more preferably 70%or more, further preferably 90% or more.

LP′ is not particularly limited by its sequence and is preferably apeptide of -(−25th)LQDLDLSELTVTSLRDTVALPENGA(−1st)- from the −25th to−1st amino acids in order from the N terminus with X in the formula(III) defined as position 1, or a peptide having homology with thispeptide. For LP′, the homology with the peptide of-LQDLDLSELTVTSLRDTVALPENGA- is preferably 50% or more, more preferably70% or more, and further preferably 90% or more.

Each of the sequences of LP and LP′ is more preferably a peptide of-(−30th)VESLDLQDLDLSELTVTSLRDTVALPENGA(−1st)- from the −30th to −1stamino acids in order from the N terminus with X defined as position 1,or a peptide having homology with this peptide, and further preferably apeptide of -(−38th)MSDITASRVESLDLQDLDLSELTVTSLRDTVALPENGA(−1st)- fromthe −38th to −1st amino acids in order from the N terminus with Xdefined as position 1, or a peptide having homology with this peptide.The homology with these peptides is preferably 50% or more, morepreferably 70% or more, and further preferably 90% or more.

LP′ preferably has a hydrophobic amino acid or a derivative thereof,more preferably leucine or an analog thereof, further preferablyleucine, at position −17 with X in the formula (III) defined as position1.

LP′ preferably has a hydrophobic amino acid or a derivative thereof,more preferably leucine or a derivative thereof, further preferablyleucine, at position −20 with X in the formula (III) defined as position1.

LP′ preferably has an acidic amino acid or a derivative thereof, morepreferably aspartic acid or a derivative thereof, further preferablyaspartic acid, at position −21 with X in the formula (III) defined asposition 1.

LP′ preferably has a hydrophobic amino acid or a derivative thereof,more preferably leucine or a derivative thereof, further preferablyleucine, at each of positions −17 and −20 with X in the formula (III)defined as position 1.

LP′ preferably has a hydrophobic amino acid or a derivative thereof ateach of positions −17 and −20 and an acidic amino acid or a derivativethereof at position −21, more preferably leucine or a derivative thereofat each of positions −17 and −20 and aspartic acid or a derivativethereof at position −21, further preferably leucine at each of positions−17 and −20 and aspartic acid at position −21, with X in the formula(III) defined as position 1.

Z in the formula (I) and the formula (II) or Z′ in the formula (III) ispresent or absent and, when present, represents a peptide consisting of1 to 100 amino acids and/or analogs thereof.

Each of Z and Z′, if present, is not particularly limited by itssequence and types of amino acids and is preferably a peptide consistingof 1 to 100 amino acids and/or analogs thereof, more preferably apeptide consisting of 1 to 50 amino acids and/or analogs thereof,further preferably a peptide consisting of 1 to 40 amino acids and/oranalogs thereof, still further preferably a peptide consisting of 3 to30 amino acids and/or analogs thereof. In Z in the formula (I) and theformula (II) or Z′ in the formula (III), the amino acid adjacent to Xcor Y is not particularly limited and is preferably an amino acid otherthan an acidic amino acid or an analog thereof.

m in the formula (I) and the formula (II) or m′ in the formula (III) isan integer selected from 2 to 40, preferably an integer of 2 or largerand 24 or smaller, more preferably an integer of 2 or larger and 20 orsmaller, further preferably an integer of 2 or larger and 16 or smaller.When each of m and m′ is an integer selected from 2 to 40, cyclizationtends to proceed smoothly by the action of macrocyclase.

Y in the formula (I) is a peptide residue consisting of four amino acidsand/or analogs thereof and contains a group represented by the formula(2) given below. The group represented by the formula (2) is a residuecorresponding to two amino acids and/or analogs thereof in the peptideresidue consisting of four amino acids and/or analogs thereof. Thepeptide residue in Y except for the group represented by the formula (2)is a residue corresponding to any two amino acids and/or analogsthereof.

wherein R¹ and R³ each represent a hydrogen or a hydrocarbon group, andB¹ represents an oxygen atom, a sulfur atom or a NH group.

Y in the formula (I) is a peptide residue consisting of four amino acidsand/or analogs thereof and preferably contains a group represented bythe following formula (2-1) or formula (2-2):

wherein R¹, R⁴ and R⁵ each represent a hydrogen atom or a hydrocarbongroup, and B¹ represents an oxygen atom, a sulfur atom or a NH group.

wherein R¹, R³ and R⁵ each represent a hydrogen atom or a hydrocarbongroup, and B¹ and B² each represent an oxygen atom, a sulfur atom or aNH group.

Y in the formula (I) is preferably a group represented by the followingformula (3):

wherein R¹, R³, R⁴ and R⁵ each independently represent a hydrogen atomor a hydrocarbon group, and B¹ and B² each represent an oxygen atom, asulfur atom or a NH group.

Y in the formula (I) is more preferably a group represented by thefollowing formula (3′):

wherein R¹, R³, R⁴ and R⁵ each independently represent a hydrogen atomor a methyl group, and B¹ and B² each represent an oxygen atom or asulfur atom, with the proviso that when B¹ and/or B² is a sulfur atom,each of R¹ and R⁵ is a hydrogen.

The group represented by the formula (3) or the formula (3′) ispreferably represented by the following formula:

Y in the formula (I) is further preferably a group represented by thefollowing formula (3″):

wherein R¹, R⁴ and R⁵ each independently represent a hydrogen atom or amethyl group, and B¹ and B² each represent an oxygen atom or a sulfuratom, with the proviso that when B¹ and/or B² is a sulfur atom, each ofR¹ and R⁵ is a hydrogen.

The group represented by the formula (3″) is preferably represented bythe following formula:

Y in the formula (I) is still further preferably a group represented bythe following formula (3-1):

Y′ in the formula (III) is a peptide consisting of four amino acidsrepresented by —Y′(10)-Y′(11)-Y′(12)-Y′(13)- and/or analogs thereof,wherein Y′(10) is an amino acid residue selected from the groupconsisting of any amino acids and analogs thereof, Y′(11) is serine,cysteine, threonine or diaminopropionic acid, or an analog thereof,Y′(12) is serine or threonine, or an analog thereof, and Y′(13) is anamino acid residue selected from the group consisting of any amino acidsand analogs thereof.

Y′(11) is preferably serine, cysteine or threonine, or an analogthereof, more preferably serine, cysteine or threonine, furtherpreferably serine.

Y′(12) is preferably serine or threonine or an analog thereof, morepreferably serine or an analog thereof, further preferably serine.

Preferably, Y′(11) is serine, cysteine or threonine, or an analogthereof, and Y′(12) is serine or an analog thereof.

Y′(10) is not particularly limited and is preferably serine orthreonine, or an analog thereof from the viewpoint of the efficiency ofthe macrocyclization reaction. Y′(13) is not particularly limited and ispreferably serine, cysteine, threonine or diaminopropionic acid, or ananalog thereof from the viewpoint of the efficiency of themacrocyclization reaction.

Y′(10) is more preferably serine or threonine, further preferablyserine.

Y′(13) is more preferably serine, cysteine or threonine, furtherpreferably cysteine.

Preferably, Y′(10) is serine or threonine or an analog thereof, Y′(11)is serine, cysteine or threonine or an analog thereof, Y′(12) is serineor an analog thereof, and Y′(13) is serine, cysteine, threonine ordiaminopropionic acid, or an analog thereof. More preferably, Y′(10) isserine or threonine, or an analog thereof, Y′(11) is serine, cysteine orthreonine or an analog thereof, Y′(12) is serine, and Y′(13) is serine,cysteine or threonine, or an analog thereof. Further preferably, Y′(10)is serine, Y′(11) is serine, Y′(12) is serine, and Y′(13) is cysteine.

For (Xa′)_(m′) in the formula (III), the efficiency of the enzymaticreaction tends to be reduced when amino acid Xa′(1) is an acidic aminoacid or an analog of the acidic amino acid.

Thus, in (Xa′)_(m′) in the formula (III), the amino acid residue Xa′(1)adjacent to X′ is preferably an amino acid other than an acidic aminoacid or an analog thereof.

For (Xa′)_(m′) in the formula (III), the efficiency of the enzymaticreaction tends to be reduced when an amino acid residue adjacent toY′(10) on an N-terminal side, i.e., amino acid residue Xa′(m′) atposition m′, is an acidic amino acid or an analog of the acidic aminoacid.

Thus, in (Xa′)_(m′) in the formula (III), the amino acid residue Xa′(m′)at position m′ is preferably an amino acid other than an acidic aminoacid or an analog thereof.

For (Xa′)_(m′) in the formula (III), the efficiency of the enzymaticreaction tends to be reduced when an amino acid residue adjacent toXa′(m′) described above on an N-terminal side, i.e., amino acid residueXa′(m′−1) at position m′−1, is an acidic amino acid or an analog of theacidic amino acid, or a basic amino acid or an analog of the basic aminoacid.

Thus, in (Xa′)_(m′) in the formula (III), the amino acid residueXa′(m′−1) at position m′−1 is preferably an amino acid other than anacidic amino acid and a basic amino acid or analogs thereof.

An amino acid adjacent to Y′(13) in Z′ in the formula (III) is any aminoacid or an analog thereof. However, the efficiency of the enzymaticreaction tends to be reduced when this amino acid is an acidic aminoacid or an analog of the acidic amino acid.

Thus, the amino acid adjacent to Y′(13) in Z′ in the formula (III) ispreferably an amino acid other than an acidic amino acid or an analogthereof.

The method for producing a peptide library according to the presentinvention employs an azole ring-forming enzyme, an α,β-unsaturated aminoacid-forming enzyme and macrocyclase.

The azole ring-forming enzyme in the method for producing a peptidelibrary according to the present invention preferably includes LazD,LazE and LazF. An enzyme having homology with each of LazD, LazE andLazF can also be used as the azole ring-forming enzyme.

Examples of the enzyme having homology with each of LazD, LazE and LazFinclude enzymes shown in Table 2 below. Two or more of these enzymes maybe used in combination. The enzyme having homology with each of LazD,LazE and LazF is also referred to as a homolog of LazD, LazE or LazF.The “homolog” means an enzyme that is an enzyme other than LazD, LazEand LazF and that enables a peptide having a formed azole ring to bedetected when an similar azole ring formation reaction as described inExamples mentioned later is performed for an arbitrary peptide with thesame concentration as that of LazD, LazE or LazF.

The homology of the homologs of LazD, LazE and LazF can be 20% or morewith respect to the alignment of LazD, LazE and LazF, respectively. Inthe present specification, the “enzyme having homology” also includes anenzyme having homology (%) of 20% or more with respect to the alignmentof the original enzyme. Found enzymes that can be used similarly toLazD, LazE or LazF on the basis of homology (%) with respect to thealignment of LazD, LazE or LazF are shown in Tables 3-1 and 3-2 (forLazD), Tables 4-1 to 4-81 (for LazE) and Tables 5A-1 to 5A-51 (forLazF). The enzymes shown in these tables have been found by homologysearch using BLAST.

In the case of using LazD, LazE and LazF for the azole ring formationaccording to the present invention, LazD and LazE form azoline, andsubsequently, LazF (a domain contained in LazF, close to C-terminalside, involved in azole ring formation) forms azole, therebyconstructing an azole ring. The azole ring-forming enzyme according tothe present invention is a group including enzymes involved in azolineformation and azole formation.

In this context, each of the homologs of LazD and LazE may be an enzymecontaining a domain involved in azoline formation and a domain involvedin an additional function together (also referred to as a bi-functionalenzyme) or may be two or more separate enzymes each containing a domaininvolved in azoline formation. LazF contains a C-terminal domaininvolved in azole formation and an N-terminal domain involved inα,β-unsaturated amino acid formation. Thus, the homolog of LazF as theazole ring-forming enzyme can be an enzyme containing at least a domaininvolved in azole formation and may be two or more separate enzymes eachcontaining a domain involved in azole formation.

As described above, the azole ring-forming enzyme is not particularlylimited as long as the enzyme contains at least domains involved inazoline formation and azole formation. The homolog of LazD, LazE or LazFpreferably contains a domain having homology of 80% or more, morepreferably homology of 85% or more, further preferably homology of 90%or more, with each of the domains involved in azoline formation andazole formation in LazD, LazE and LazF.

The α,β-unsaturated amino acid-forming enzyme in the method forproducing a peptide library according to the present inventionpreferably includes LazB and LazF. An enzyme having homology with eachof LazB and LazF can also be used as the α,β-unsaturated aminoacid-forming enzyme.

Examples of the enzyme having homology with each of LazB and LazFinclude enzymes shown in Tables 6-1 to 6-13 (for LazB) and Tables 5B-1to 5B-32 (for LazF) below. Two or more of these enzymes may be used incombination.

The enzyme having homology with each of LazB and LazF is also referredto as a homolog of LazB or LazF. The “homolog” means an enzyme that isan enzyme other than LazB and LazF and that enables a peptide having aformed α,β-unsaturated amino acid to be detected when an similarα,β-unsaturated amino acid formation reaction as described in Examplesmentioned later is performed for an arbitrary peptide with the sameconcentration as that of LazB or LazF.

The homology of the homologs of LazB and LazF can be 20% or more withLazB and LazF, respectively.

In the case of using LazB and LazF for the α,β-unsaturated amino acidformation according to the present invention, LazB performsglutamylation, and subsequently, LazF (a domain of LazF, close toN-terminal side, involved in α,β-unsaturated amino acid formation)causes glutamate elimination, thereby constructing an α,β-unsaturatedamino acid. The α,β-unsaturated amino acid-forming enzyme according tothe present invention is a group including enzymes involved inglutamylation and glutamate elimination.

In this context, each of the homologs of LazB and LazF may be an enzymecontaining a domain involved in glutamylation or glutamate eliminationand a domain involved in an additional function together (also referredto as a bi-functional enzyme) or may be two or more separate enzymeseach containing a domain involved in glutamylation or glutamateelimination.

As described above, the α,β-unsaturated amino acid-forming enzyme is notparticularly limited as long as the enzyme contains at least domainsinvolved in glutamylation and glutamate elimination. The homologs ofLazB and LazF preferably contain a domain having homology of 80% ormore, more preferably homology of 85% or more, further preferablyhomology of 90% or more, with the domains involved in glutamylation andglutamate elimination in LazB and LazF, respectively.

The macrocyclase in the method for producing a peptide library accordingto the present invention preferably includes LazC. An enzyme havinghomology with LazC can also be used as the macrocyclase.

Examples of the enzyme having homology with LazC include enzymes shownin Tables 7-1 to 7-6 below. Two or more of these enzymes may be used incombination.

The enzyme having homology with LazC is also referred to as a homolog ofLazC. The “homolog” means an enzyme that is an enzyme other than LazCand that enables a peptide having a formed macrocycle to be detectedwhen an similar macrocyclization reaction as described in Examplesmentioned later is performed for an arbitrary peptide with the sameconcentration as that of LazC.

The homology of the homolog of LazC can be 20% or more with LazC.

The macrocyclase is not particularly limited as long as the enzymecontains at least a domain involved in macrocyclization. The homolog ofLazC may be an enzyme containing a domain involved in macrocyclizationand a domain involved in an additional function together (also referredto as a bi-functional enzyme) or may be two or more separate enzymeseach containing a domain involved in macrocyclization. The homolog ofLazC preferably contains a domain having homology of 80% or more, morepreferably homology of 85% or more, further preferably homology of 90%or more, with the domain involved in macrocyclization in LazC.

In the present specification, the phrase “having homology of Y % or morewith the amino acid sequence represented by SEQ ID NO: X” means thatwhen two polypeptides are aligned so as to match their amino acidsequences to the maximum, the ratio of the number of common amino acidresidues to the total number of amino acids shown in SEQ ID NO: X is Y %or more.

TABLE 2 Dha/Dhb formation Azole cyclization DehydroalanineDehydroalanine Name of specific Azoline formation formation compound tobe cyclization Azoline cyclization Azoline oxidation (glutamic acid(glutamate synthesized (docking) (cyclodehydration) (azole cyclization)addition) elimination) Macrocyclization Thipeptide Lactazole LazD LazELazF C-ter LazB LazF N-ter LazC Micrococcin = TclI TclJ TclN TclK TclLTclM Thiocillin Thiocillin = TelI TclJ TclN TclK TclL TclM MicrococcinBerninamycin BerE2 BerE2 BerE1 BerB BerC BerD BerG1 BerG2 BerE2Thiomuracin TbtF TbtG TbtE TbtB TbtC TbtD Thiomuracin TpdF TpdG TpdETpdB TpdC TpdD GE2270 PbtF PbtG PbtE PbtB PbtC PbtD GE2270 TpdF TpdGTpdE TpdB TpdC TpdD Nosiheptide NosH NosG NosF NosE NosD NosOThiostrepton TsrN TsrO TsrM TsrJ TsrK TsrL TsrS Siomycin SioN SioO SioMSioJ SioK SioL SioS TP-1161 TpdG TpaH TpaE TpaL TpaK TpaB TpaC TpaF TpaDThiocillin TclI TclJ TclN TclK TclL TclM GE37468 GetH GetI GetG GetDGetE GetF GetK Cyclothiazomycin CltD CltB CltC CltE CltF CltD CltG LAPsGoadsporin GodD GodD GodE GodF GodG Plantazolicin BamC BamD BamBcyanobactin Patellamide PatD PatG unnamed LynD LynG cyanobactinTrichamide TriD TriG Tenuecyclamide TenD TenG Trunkamide TruD TruGLantipeptide Nisin NisB NisB NAI-107 MibB MibB Name of specific compoundto be synthesized Producing microbe Reference Thipeptide LactazoleStreptomyces lactacystinaeus OM-6519 Chem. Biol. 21 (5), 679-688 (2014)Micrococcin = Staphylococcus epidermidis strain 115 J. Bacteriol. 2016,198, 2431-2438 Thiocillin Thiocillin = Bacillus cereus ATCC 14579 PNAS,2009. 106 (8) 2549-2553 Micrococcin Berninamycin Streptomyces bernensisUC 5144 PNAS, 2013. 110 (21) 8483-8488 Thiomuracin Thermobispora bisporaDSM 43833 J Am Chem Soc. 2015 137(51): 16012-16015. ThiomuracinNonomuraea Bp3714-39 J Am Chem Soc. 2009; 131(16): 5946-55. GE2270Planobispora rosea ATCC53733 J Am Chem Soc. 2015 137(51): 16012-16015.GE2270 Nonomuraea strain WU8817 J Am Chem Soc. 2009; 131(16): 5946-55.Nosiheptide Streptomyces actuosus ATCC 25421 ACS Chem. Biol., 2009, 4(10), pp 855-864 Thiostrepton Streptomyces laurentii ATCC 31255Chemistry & Biology, 16, 2009, 141-147 Siomycin Streptomyces sioyaensisATCC 13989 Chemistry & Biology, 16, 2009, 141-147 TP-1161 Nocardiopsissp. TFS65-07 Appl. Environ. Microbiol. 76 (21), 7093-7101 (2010)Thiocillin Bacillus cereus ATCC 14579 PNAS, 2009. 106 (8) 2549-2553GE37468 Streptomyces ATCC 55365 PNAS, 2011. 108 (32) 13053-13058Cyclothiazomycin Streptomyces hygroscopicus 10-22 Appl. Environ.Microbiol. 76 (7), 2335-2344 (2010) LAPs Goadsporin Streptomyces sp.Tp-A0584 Microbiology 151, 3923-3933 (2005) Plantazolicin Bacillusmethylotrophicus FZB42 ACS Chem. Biol., 2016, 11, 2232-2243 cyanobactinPatellamide PNAS, 2005. 102 (20) 7315-7320 unnamed Nat Chem Biol. 2008June; 4(6): 341-343. cyanobactin Trichamide Nat Chem Biol. 2008 June;4(6): 341-343. Tenuecyclamide Nat Chem Biol. 2008 June; 4(6): 341-343.Trunkamide Prochloron didemni Nat Chem Biol. 2008 June; 4(6): 341-343.Lantipeptide Nisin Lactococcus lactis 6F3 Nature 517, 509-512 (2015)NAI-107 Microbispora corallina PNAS, 2010. 107 (30) 13461-13466

TABLE 3-1 Max Total Query E Description score score cover value IdentAccession hypothetical protein [Streptomyces lactacystinaeus] 1033 1033100%  0 100%  BAO57437.1 hypothetical protein [Streptomyces sp. CB02056]577 577 100%  0 81% WP_074003107.1 hypothetical protein [Streptomycessp. 1331.2] 550 550 98% 0 71% WP_097239703.1 hypothetical protein[Streptomyces sp. NRRL S-350] 532 532 99%  2.00E−180 67% WP_051709135.1hypothetical protein [Streptomyces sp. TLI_053] 447 447 98%  1.00E−14758% WP_093858545.1 hypothetical protein [Streptomyces sp. MJM8645] 447447 98%  1.00E−147 62% WP_063345886.1 hypothetical protein [Streptomycessp. 61] 390 390 98%  1.00E−125 52% WP_099888077.1 hypothetical protein[Streptomyces sp. NRRL S-87] 381 381 97%  4.00E−121 49% WP_051795Q70.1hypothetical protein [Actinobacteria bacterium OV450] 375 375 98% 2.00E−119 50% WP_054221079.1 hypothetical protein [Streptomyces sp.NRRL S-1022] 369 369 98%  2.00E−116 47% WP_051789975.1 hypotheticalprotein [Streptomyces sp. XY332] 367 367 98%  2.00E−116 53%WP_053790559.1 hypothetical protein [Streptomyces sp. 1] 363 363 99% 8.00E−116 60% WP_099897192.1 hypothetical protein [Streptomycescellostaticus] 357 429 96%  4.00E−111 49% WP_067007687.1 hypotheticalprotein [Streptomyces sp. NRRL B-3648] 348 348 98%  5.00E−108 46%WP_053707099.1 hypothetical protein [Streptomyces katrae] 338 338 98% 1.00E−105 53% WP_051832197.1 hypothetical protein [Streptomyces roseus]337 337 98%  1.00E−104 48% WP_048479857.1 hypothetical protein[Streptomyces glaucescens] 320 320 85% 5.00E−99 51% WP_086741346.1hypothetical protein OQI_38750 [Streptomyces pharetrae CZA14] 308 30872% 9.00E−96 53% OSZ55401.1 hypothetical protein [Streptomyces sp. NRRLF-2747] 301 301 87% 1.00E−91 51% WP_051806535.1 hypothetical protein[Streptomyces albus] 280 280 98% 1.00E−82 43% WP_050505355.1hypothetical protein [Streptomyces sp. ScaeMP-e48] 278 278 97% 7.00E−8243% WP_099127122.1 hypothetical protein [Streptomyces alboverticillatus]275 275 97% 2.00E−81 45% WP_086573079.1 hypothetical protein[Streptomyces alboviridis] 276 276 97% 6.00E−81 43% WP_032759558.1hypothetical protein [Streptomyces glaucescens] 275 275 97% 1.00E−80 43%WP_052413540.1 hypothetical protein [Streptomyces sp. CFMR 7] 271 27197% 2.00E−79 43% WP_053557898.1 hypothetical protein [Kitasatosporaazatica] 268 268 98% 9.00E−79 46% WP_035839234.1 hypothetical protein[Streptomyces sp. WM4235] 268 268 96% 5.00E−78 42% WP_053684586.1hypothetical protein [Streptomyces sp. PgraA7] 266 266 98% 1.00E−77 43%WP_088573439.1 hypothetical protein BV881_27315 [Streptomyces sp. ZL-24]265 265 97% 8.00E−77 42% POG44347.1 hypothetical protein [Kitasatosporasp. MMS16-BH015] 259 259 91% 4.00E−75 45% WP_104819510.1 hypotheticalprotein [Streptomyces sp. S8] 258 258 97% 2.00E−74 42% WP_084990319.1hypothetical protein [Streptomyces sp. CcalMP-8W] 257 257 97% 8.00E−7442% WP_018487808.1 hypothetical protein [Streptomyces cyaneofuscatus]256 256 99% 9.00E−74 44% WP_030565277.1 hypothetical protein[Streptomyces sp. ScaeMP-e83] 257 257 97% 1.00E−73 40% WP_093688171.1hypothetical protein [Streptomyces luridiscabiei] 255 255 97% 3.00E−7343% WP_055558883.1 hypothetical protein [Streptomyces sp. NRRL S-623]254 254 97% 5.00E−73 42% WP_031125357.1 hypothetical protein[Streptomyces sp. SolWspMP-sol2th] 252 252 97% 6.00E−72 42%WP_028417712.1 hypothetical protein [Streptomyces sp. AmelKG-D3] 246 24697% 7.00E−70 42% WP_099219967.1 hypothetical protein [Streptomyces sp.CB00316] 245 245 98% 2.00E−69 42% WP_073882412.1 hypothetical protein[Streptomyces sp. 2R] 244 244 97% 3.00E−69 42% WP_094213839.1hypothetical protein C6W96_33530 [Streptomyces sp. CS149] 244 244 98%4.00E−69 43% PSK68234.1 hypothetical protein [Streptomyces sp.DvalAA-19] 242 242 97% 3.00E−68 43% WP_093751378.1 hypothetical protein[Streptomyces sp. Sge12] 241 241 92% 7.00E−68 43% WP_081521136.1hypothetical protein [Streptomyces sp. Tue 6075] 239 239 98% 3.00E−6743% WP_075262962.1 hypothetical protein [Streptomyces sp. ScaeMP-e10]238 238 99% 8.00E−67 42% WP_018512748.1 hypothetical protein[Streptomyces nanshensis] 231 231 81% 3.00E−65 44% WP_070200360.1hypothetical protein [Streptomyces sp. rh34] 229 229 77% 2.00E−64 41%WP_097871222.1 hypothetical protein [Streptomyces sp. WM6372] 231 23158% 2.00E−64 50% WP_053694255.1 hypothetical protein [Streptomyceslavendulae] 225 225 92% 1.00E−62 44% WP_100661069.1

TABLE 3-2 Max Total Query E Description score score cover value IdentAccession hypothetical protein [Streptomyces sp. TSRI0107] 224 314 95%2.00E−61 43% WP_073939586.1 hypothetical protein [Streptomyces sp. NRRLF-4335] 218 218 92% 1.00E−59 42% WP_052876897.1 hypothetical protein[Streptomyces sp. st77] 214 214 78% 2.00E−59 44% WP_098899758.1hypothetical protein [Streptomyces griseochromogenes] 215 368 93%5.00E−58 51% WP_067306575.1 hypothetical protein [Streptomyces sp. NRRLF-2580] 209 209 98% 2.00E−56 42% WP_051838076.1 hypothetical protein[Streptomyces sp. CB00455] 202 202 78% 3.00E−53 41% WP_073917949.1hypothetical protein [Streptomyces sp. NRRL. S-515] 191 191 98% 2.00E−4941% WP_051782675.1 hypothetical protein [Streptomyces sp. WM6368] 186186 73% 4.00E−49 42% WP_053702090.1 hypothetical protein [Streptomycesvirginiae] 185 185 82% 6.00E−48 41% WP_053168739.1 hypothetical proteinGA0115280_110133 [Streptomyces sp. 181 181 68% 1.00E−47 44% SGF75265.1Cmuel-A718b] hypothetical protein [Streptomyces katrae] 186 186 78%2.00E−47 45% WP_079424624.1 hypothetical protein [Streptomyces sp.Cmuel-A718b] 181 181 68% 2.00E−47 44% WP_093442406.1 hypotheticalprotein [Amycolatopsis xylanica] 176 176 64% 1.00E−45 44% WP_091295326.1collagen-like protein [Streptomyces sp. Ag109_G2-1] 180 180 78% 3.00E−4545% WP_100865208.1 hypothetical protein [Streptomyces somaliensis] 178178 96% 6.00E−45 42% WP_010472145.1 hypothetical protein [Hamadaeatsunoensis] 156 156 92% 9.00E−38 33% WP_027344872.1 hypothetical protein[Streptomyces sp. NRRL S-575] 138 138 73% 9.00E−32 41% WP_078919492.1hypothetical protein AN221_07435 [Streptomyces nanshensis] 134 134 45%2.00E−31 46% OEV21369.1 hypothetical protein BOI31_12545 [Saccharothrixcarnea] 135 135 75% 4.00E−30 35% PSL47138.1 hypothetical protein[Saccharothrix sp. NRRL B-16348] 133 133 92% 1.00E−29 34% WP_053719226.1hypothetical protein [Microtetraspora malaysiensis] 126 126 73% 4.00E−2733% WP_067140202.1 hypothetical protein [Saccharothrix sp. NRRL B-16348]117 117 78% 4.00E−24 36% WP_053719745.1 hypothetical proteinSAMN04488074_10149 [Lentzea violacea] 115 115 92% 1.00E−23 33%SDI92398.1 hypothetical protein SAMN05216195_103210 111 111 92% 2.00E−2232% SEQ85151.1 [Lentzea flaviverrucosa] hypothetical protein[Microtetraspora malaysiensis] 111 111 77% 3.00E−22 34% WP_067140224.1hypothetical protein [Actinosynnema mirum] 110 110 92% 4.00E−22 35%WP_015802629.1 hypothetical protein SAMN05660733_02664 107 107 92%3.00E−21 33% SMC91656.1 [Lentzea albidocapillata] hypothetical protein[Actinosynnema pretiosum] 107 107 75% 4.00E−21 36% WP_096495003.1hypothetical protein VR44_27320 [Streptomyces katrae] 94.7 94.7 28%6.00E−19 49% KJY27540.1 hypothetical protein SAMN04488564_1021002 94.494.4 92% 1.00E−16 29% SFR06408.1 [Lentzea waywayandensis] hypotheticalprotein [Streptomyces paucisporeus] 90.9 90.9 58% 2.00E−15 37%WP_073498162.1 hypothetical protein B5180_35195 [Streptomyces sp. BF-3]82.4 82.4 22% 1.00E−14 50% OSC61991.1 hypothetical proteinGA0115280_11522 [Streptomyces sp. 82.4 82.4 22% 1.00E−14 50% SCF83876.1Cmuel-A718b] hypothetical protein GA0115253_105541 [Streptomyces sp.82.4 82.4 28% 3.00E−14 47% SCE45950.1 Termitarium-T10T-6] hypotheticalprotein SAMN05216188_108124 82 132 82% 1.00E−12 34% SER15500.1[Lechevalieria xinjiangensis] hypothetical protein [Amycolatopsisorientalis] 80.9 80.9 88% 2.00E−12 32% WP_044856427.1 hypotheticalprotein PL81_15150 [Streptomyces sp. RSD-27] 70.1 70.1 15% 3.00E−11 58%KIF05079.1 hypothetical protein [Cryptosporangium aurantiacum] 73.9 73.965% 4.00E−10 34% WP_073265669.1 hypothetical protein GA0115253_104491[Streptomyces sp. 65.9 65.9 34% 2.00E−08 37% SCE34462.1Termitarium-T10T-6] hypothetical protein B5180_26600 [Streptomyces sp.BF-3] 63.9 63.9 29% 4.00E−08 42% OSC69702.1 hypothetical proteinOQI_37530 [Streptomyces pharetrae 56.2 56.2 13% 1.00E−06 52% OSZ55612.1CZA14] hypothetical protein BOI31_12617 [Saccharothrix carnea] 60.1 60.114% 7.00E−06 43% PSL45986.1 hypothetical protein AN221_07450[Streptomyces nanshensis] 48.1 48.1 13% 0.003 43% OEV21371.1hypothetical protein [Amycolatopsis orientalis] 48.9 48.9 44% 0.021 34%WP_037311642.1 hypothetical protein PL81_32215 [Streptomyces sp. RSD-27]42 42  6% 0.16 54% KIF02043.1 aldehyde dehydrogenase [Streptomycesniveus NCIMB 11891] 40.8 40.8 32% 8.1 36% EST21127.1 aldehydedehydrogenase (NADP(+)) [Streptomyces sp. Wb2n-11] 40.8 40.8 20% 8.7 36%WP_093801440.1 aldehyde dehydrogenase (NADP(+)) [Streptomyces niveus]40.8 40.8 32% 9.4 36% WP_078494390.1

TABLE 4-1 Max Total Query E Description score score cover value IdnetAccession

% 0

%

.1

% 0

%

.1

% 0

%

.1

% 0

%

.1

% 0

%

.1

% 0

%

.1

% 0

%

.1

% 0

%

.1

% 0

%

.1

% 0

%

.1

% 0

%

.1

% 0

%

.1

% 0

%

.1

% 0

%

.1

% 0

%

.1

% 0

%

.1

% 0

%

.1

% 0

%

.1

% 0

%

.1

% 0

%

.1

% 0

%

.1

% 0

%

.1

% 0

%

.1

% 0

%

.1

% 0

%

.1

% 0

%

.1

% 0

%

.1

% 0

%

.1

% 0

%

.1

% 0

%

.1

% 0

%

.1

% 0

%

.1

% 0

%

.1

% 0

%

.1

% 0

%

.1

% 0

%

.1

% 0

%

.1

% 0

%

.1

% 0

%

.1

% 0

%

.1

% 0

%

.1

% 0

%

.1

% 0

%

.1

% 0

%

.1

% 0

%

.1

% 0

%

.1

% 0

%

.1

% 0

%

.1

% 0

%

.1

% 0

%

.1

% 0

%

.1

% 0

%

.1

% 0

%

.1

% 0

%

.1

% 0

%

.1

% 0

%

.1

% 0

%

.1

% 0

%

.1

% 0

%

.1

indicates data missing or illegible when filed

TABLE 4-2 Max Total Query E Description score score cover value IdnetAccession

% 0

%

.1

% 0

%

.1

% 0

%

.1

% 0

%

.1

% 0

%

.1

% 0

%

.1

% 0

%

.1

% 0

%

.1

% 0

%

.1

% 0

%

.1

% 0

%

.1

% 0

%

.1

% 0

%

.1

% 0

%

.1

% 0

%

.1

% 0

%

.1

% 0

%

.1

% 0

%

.1

% 0

%

.1

% 0

%

.1

% 0

%

.1

% 0

%

.1

% 0

%

.1

% 0

%

.1

% 0

%

.1

% 0

%

.1

% 0

%

.1

% 0

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

indicates data missing or illegible when filed

TABLE 4-3 Max Total Query E Description score score cover value IdentAccession

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

indicates data missing or illegible when filed

TABLE 4-4 Max Total Query E Description score score cover value IdentAccession

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

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%

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%

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%

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%

.1

indicates data missing or illegible when filed

TABLE 4-5 Max Total Query E Description score score cover value IdentAccession

%

E−

%

.1

%

E−

%

.1

%

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%

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%

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%

.1

%

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%

.1

indicates data missing or illegible when filed

TABLE 4-6 Max Total Query E Max Description score score cover valuescore Accession

%

E−

%

.1

%

E−

%

.1

%

E−

%

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%

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%

.1

%

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%

.1

indicates data missing or illegible when filed

TABLE 4-7 Max Total Query E Max Description score score cover valuescore Accession

%

E−

%

.1

%

E−

%

.1

%

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%

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%

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%

.1

%

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%

.1

indicates data missing or illegible when filed

TABLE 4-8 Max Total Query E Description score score cover value IdentAccession

%

E−

%

.1

%

E−

%

.1

%

E−

%

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E−

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%

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%

.1

%

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%

.1

indicates data missing or illegible when filed

TABLE 4-9 Max Total Query E Description score score cover value IdentAccession

%

E−

%

.1

%

E−

%

.1

%

E−

%

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%

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%

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%

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%

.1

%

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%

.1

indicates data missing or illegible when filed

TABLE 4-10 Max Total Query E Description score score cover value IdentAccession

%

E−

%

.1

%

E−

%

.1

%

E−

%

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%

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%

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%

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%

.1

%

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%

.1

indicates data missing or illegible when filed

TABLE 4-11 Max Total Query E Description score score cover value IdentAccession

%

E−

%

.1

%

E−

%

.1

%

E−

%

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%

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%

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%

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%

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%

.1

indicates data missing or illegible when filed

TABLE 4-12 Max Total Query E Description score score cover value IdentAccession

%

E−

%

.1

%

E−

%

.1

%

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%

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%

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%

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%

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%

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indicates data missing or illegible when filed

TABLE 4-13 Max Total Query E Description score score cover value IdentAccession

%

E−

%

.1

%

E−

%

.1

%

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%

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%

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%

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%

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%

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%

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indicates data missing or illegible when filed

TABLE 4-14 Max Total Query E Description score score cover value IdentAccession

%

E−

%

.1

%

E−

%

.1

%

E−

%

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%

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%

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%

.1

%

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%

.1

indicates data missing or illegible when filed

TABLE 4-15 Max Total Query E Description score score cover value IdentAccession

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

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%

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%

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%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

indicates data missing or illegible when filed

TABLE 4-16 Max Total Query E Description score score cover value IdentAccession

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

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%

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%

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%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

indicates data missing or illegible when filed

TABLE 4-17 Max Total Query E Description score score cover value IdentAccession

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

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%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

indicates data missing or illegible when filed

TABLE 4-18 Max Total Query E Description score score cover value IdentAccession

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

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%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

indicates data missing or illegible when filed

TABLE 4-19 Max Total Query E Description score score cover value IdentAccession

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

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%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

indicates data missing or illegible when filed

TABLE 4-20 Max Total Query E Description score score cover value IdentAccession

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

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%

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%

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E−

%

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%

E−

%

.1

%

E−

%

.1

indicates data missing or illegible when filed

TABLE 4-21 Max Total Query E Description score score cover value IdentAccession

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

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%

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%

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%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

indicates data missing or illegible when filed

TABLE 4-22 Max Total Query E Description score score cover value IdentAccession

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

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%

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%

.1

%

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%

.1

indicates data missing or illegible when filed

TABLE 4-23 Max Total Query E Description score score cover value IdentAccession

%

E−

%

.1

%

E−

%

.1

%

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%

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%

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%

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%

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%

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%

.1

indicates data missing or illegible when filed

TABLE 4-24 Max Total Query E Description score score cover value IdentAccession

%

E−

%

.1

%

E−

%

.1

%

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%

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%

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%

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%

.1

indicates data missing or illegible when filed

TABLE 4-25 Max Total Query E Description score score cover value IdentAccession

%

E−

%

.1

%

E−

%

.1

%

E−

%

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%

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%

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%

.1

%

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%

.1

indicates data missing or illegible when filed

TABLE 4-26 Max Total Query E Description score score cover value IdentAccession

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

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%

E−

%

.1

%

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%

.1

indicates data missing or illegible when filed

TABLE 4-27 Max Total Query E Description score score cover value IdentAccession

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

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%

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%

.1

%

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%

.1

indicates data missing or illegible when filed

TABLE 4-28 Max Total Query E Description score score cover value IdentAccession

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

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%

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%

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%

.1

indicates data missing or illegible when filed

TABLE 4-29 Max Total Query E Description score score cover value IdentAccession

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

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%

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%

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%

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indicates data missing or illegible when filed

TABLE 4-30 Max Total Query E Description score score cover value IdentAccession

%

E−

%

.1

%

E−

%

.1

%

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%

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%

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%

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indicates data missing or illegible when filed

TABLE 4-31 Max Total Query E Description score score cover value IdentAccession

%

E−

%

.1

%

E−

%

.1

%

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%

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%

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indicates data missing or illegible when filed

TABLE 4-32 Max Total Query E Description score score cover value IdentAccession

%

E−

%

.1

%

E−

%

.1

%

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%

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%

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%

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indicates data missing or illegible when filed

TABLE 4-33 Max Total Query E Description score score cover value IdentAccession

%

E−

%

.1

%

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%

.1

%

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%

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%

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%

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%

.1

indicates data missing or illegible when filed

TABLE 4-34 Max Total Query E Description score score cover value IdentAccession

%

E−

%

.1

%

E−

%

.1

%

E−

%

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%

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%

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%

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%

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%

.1

indicates data missing or illegible when filed

TABLE 4-35 Max Total Query E Description score score cover value IdentAccession

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

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%

E−

%

.1

%

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%

.1

indicates data missing or illegible when filed

TABLE 4-36 Max Total Query E Description score score cover value IdentAccession

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

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%

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%

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%

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%

.1

indicates data missing or illegible when filed

TABLE 4-37 Max Total Query E Description score score cover value IdentAccession

%

E−

%

.1

%

E−

%

.1

%

E−

%

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%

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%

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%

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%

.1

indicates data missing or illegible when filed

TABLE 4-38 Max Total Query E Description score score cover value IdentAccession

%

E−

%

.1

%

E−

%

.1

%

E−

%

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%

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%

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%

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%

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%

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indicates data missing or illegible when filed

TABLE 4-39 Max Total Query E Description score score cover value IdentAccession

%

E−

%

.1

%

E−

%

.1

%

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%

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%

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%

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%

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indicates data missing or illegible when filed

TABLE 4-40 Max Total Query E Description score score cover value IdentAccession

%

E−

%

.1

%

E−

%

.1

%

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%

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%

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indicates data missing or illegible when filed

TABLE 4-41 Max Total Query E Description score score cover value IdentAccession

%

E−

%

.1

%

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%

.1

%

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indicates data missing or illegible when filed

TABLE 4-42 Max Total Query E Description score score cover value IdentAccession

%

E−

%

.1

%

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%

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%

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%

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%

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%

.1

indicates data missing or illegible when filed

TABLE 4-43 Max Total Query E Description score score cover value IdentAccession

%

E−

%

.1

%

E−

%

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%

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indicates data missing or illegible when filed

TABLE 4-44 Max Total Query E Description score score cover value IdentAccession

%

E−

%

.1

%

E−

%

.1

%

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%

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%

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%

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%

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indicates data missing or illegible when filed

TABLE 4-45 Max Total Query E Description score score cover value IdentAccession

%

E−

%

.1

%

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%

.1

%

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%

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%

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%

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%

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indicates data missing or illegible when filed

TABLE 4-46 Max Total Query E Description score score cover value IdentAccession

%

E−

%

.1

%

E−

%

.1

%

E−

%

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%

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%

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%

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indicates data missing or illegible when filed

TABLE 4-47 Max Total Query E Description score score cover value IdentAccession

%

E−

%

.1

%

E−

%

.1

%

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%

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%

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%

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indicates data missing or illegible when filed

TABLE 4-48 Max Total Query E Description score score cover value IdentAccession

%

E−

%

.1

%

E−

%

.1

%

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%

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%

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indicates data missing or illegible when filed

TABLE 4-49 Max Total Query E Description score score cover value IdentAccession

%

E−

%

.1

%

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%

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%

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indicates data missing or illegible when filed

TABLE 4-50 Max Total Query E Description score score cover value IdentAccession

%

E−

%

.1

%

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%

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%

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indicates data missing or illegible when filed

TABLE 4-51 Max Total Query E Description score score cover value IdentAccession

%

E−

%

.1

%

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%

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%

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%

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indicates data missing or illegible when filed

TABLE 4-52 Max Total Query E Description score score cover value IdentAccession

%

E−

%

.1

%

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%

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%

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%

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%

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%

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%

.1

indicates data missing or illegible when filed

TABLE 4-53 Max Total Query E Description score score cover value IdentAccession

%

E−

%

.1

%

E−

%

.1

%

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%

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%

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%

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%

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%

.1

indicates data missing or illegible when filed

TABLE 4-54 Max Total Query E Description score score cover value IdentAccession

%

E−

%

.1

%

E−

%

.1

%

E−

%

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%

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%

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%

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%

.1

indicates data missing or illegible when filed

TABLE 4-55 Max Total Query E Description score score cover value IdentAccession

%

E−

%

.1

%

E−

%

.1

%

E−

%

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%

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%

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%

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%

.1

indicates data missing or illegible when filed

TABLE 4-56 Max Total Query E Description score score cover value IdentAccession

%

E−

%

.1

%

E−

%

.1

%

E−

%

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%

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%

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%

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%

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%

.1

indicates data missing or illegible when filed

TABLE 4-57 Max Total Query E Description score score cover value IdentAccession

%

E−

%

.1

%

E−

%

.1

%

E−

%

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%

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%

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%

.1

%

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%

.1

indicates data missing or illegible when filed

TABLE 4-58 Max Total Query E Description score score cover value IdentAccession

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

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%

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%

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%

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%

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indicates data missing or illegible when filed

TABLE 4-59 Max Total Query E Description score score cover value IdentAccession

%

E−

%

.1

%

E−

%

.1

%

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%

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%

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%

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%

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%

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indicates data missing or illegible when filed

TABLE 4-60 Max Total Query E Description score score cover value IdentAccession

%

E−

%

.1

%

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%

.1

%

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%

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%

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indicates data missing or illegible when filed

TABLE 4-61 Max Total Query E Description score score cover value IdentAccession

%

E−

%

.1

%

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%

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%

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%

.1

%

E−

%

.1

%

E−

%

.1

indicates data missing or illegible when filed

TABLE 4-62 Max Total Query E Description score score cover value IdentAccession

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

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%

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%

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%

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%

.1

indicates data missing or illegible when filed

TABLE 4-63 Max Total Query E Description score score cover value IdentAccession

%

E−

%

.1

%

E−

%

.1

%

E−

%

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%

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E−

%

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%

E−

%

.1

%

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%

.1

indicates data missing or illegible when filed

TABLE 4-64 Max Total Query E Description score score cover value IdentAccession

%

E−

%

.1

%

E−

%

.1

%

E−

%

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%

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%

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%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

indicates data missing or illegible when filed

TABLE 4-65 Max Total Query E Description score score cover value IdentAccession

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

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.1

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%

.2

%

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%

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%

E−

%

.1

%

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%

.1

indicates data missing or illegible when filed

TABLE 4-66 Max Total Query E Description score score cover value IdentAccession

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

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%

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%

E−

%

.1

%

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%

.1

indicates data missing or illegible when filed

TABLE 4-67 Max Total Query E Description score score cover value IdentAccession

%

E−

%

.1

%

E−

%

.1

%

E−

%

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%

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%

E−

%

.1

%

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%

.1

indicates data missing or illegible when filed

TABLE 4-68 Max Total Query E Description score score cover value IdentAccession

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

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E−

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%

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%

.1

%

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%

.1

indicates data missing or illegible when filed

TABLE 4-69 Max Total Query E Description score score cover value IdentAccession

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

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%

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%

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%

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%

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indicates data missing or illegible when filed

TABLE 4-70 Max Total Query E Description score score cover value IdentAccession

%

E−

%

.1

%

E−

%

.1

%

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%

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%

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%

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%

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%

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%

.1

indicates data missing or illegible when filed

TABLE 4-71 Max Total Query E Description score score cover value IdentAccession

%

E−

%

.1

%

E−

%

.1

%

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%

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%

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%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

indicates data missing or illegible when filed

TABLE 4-72 Max Total Query E Description score score cover value IdentAccession

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

indicates data missing or illegible when filed

TABLE 4-73 Max Total Query E Description score score cover value IdentAccession

%

E−

%

.1

%

E−

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

indicates data missing or illegible when filed

TABLE 4-74 Max Total Query E Description score score cover value IdentAccession

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

indicates data missing or illegible when filed

TABLE 4-75 Max Total Query E Description score score cover value IdentAccession

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.2

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

indicates data missing or illegible when filed

TABLE 4-76 Max Total Query E Description score score cover value IdentAccession

%

%

.2

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

indicates data missing or illegible when filed

TABLE 4-77 Max Total Query E Description score score cover value IdentAccession

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

indicates data missing or illegible when filed

TABLE 4-78 Max Total Query E Description score score cover value IdentAccession

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.3

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

indicates data missing or illegible when filed

TABLE 4-79 Max Total Query E Description score score cover value IdentAccession

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

indicates data missing or illegible when filed

TABLE 4-80 Max Total Query E Description score score cover value IdentAccession

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

indicates data missing or illegible when filed

TABLE 4-81 Max Total Query E Description score score cover value IdentAccession hypothetical protein [Streptomyces milbemycinicus] 41.6 41.620% 5.7 29% WP_086860705.1 hypothetical protein IG7_03413 [Bacilluscereus HuA2-4] 41.6 41.6 10% 5.7 35% EJQ68438.1 collagen-like repeatpreface domain-containing protein [Bacillus cereus] 41.6 41.6 11% 5.836% WP_074602647.1 hypothetical protein [Streptomyces bingchenggensis]41.6 41.6 20% 5.9 29% WP_014176997.1 hypothetical protein [Bacillusluciferensis] 41.6 41.6 10% 6.7 43% WP_084006987.1 collagen-like protein[Bacillus wiedmannii] 41.2 41.2 11% 6.7 33% PGD36547.1 collagen-likerepeat preface domain-containing protein [Bacillus cereus] 41.6 41.6 11%7.1 36% WP_080444755.1 Leu/Phe-tRNA protein transferase, putative[Plasmodium ovale curtisi] 41.6 41.6  9% 7.1 43% SBS81493.1 hypotheticalprotein [Ensifer adhaerens] 41.2 41.2 28% 7.1 27% WP_053253147.1collagen-like protein [Bacillus cereus] 41.8 82.8 11% 7.1 36%WP_088859614.1 hypothetical protein MBOVJF4278_00811 [Mycoplasma bovis]40 40 21% 7.1 20% SBO46572.1 BclB domain-containing protein[Viridibacillus sp. OK051] 41.2 41.2 15% 7.4 33% WP_100796218.1KHDC3-like protein [Trichechus manatus latirostris] 39.7 39.7 11% 7.441% XP_004381444.1 hypothetical protein [Agrococcus casei] 39.7 39.7 10%7.5 36% WP_086993089.1 VspE [Mycoplasma bovis] 40 40 21% 7.7 21%AMW25311.1 hypothetical protein [Herbidospora cretacea] 41.2 41.2 56%7.9 28% WP_030455410.1 protein qua-1-like [Varroa jacobsoni] 41.8 41.8 9% 8 38% XP_022689758.1 Serine/threonine protein kinase [Actinoplanesderwentensis] 41.6 41.6 10% 8.3 39% SDT76483.1 collagen-like protein[Bacillus cereus] 41.6 41.6 11% 8.4 34% WP_063553418.1 collagen-liketriple helix repeat protein, glycine-rich [Bacillus cereus E33L] 41.241.2 12% 8.5 33% AAU16795.1 collagen-like protein [Bacillus wiedmannii]40.4 40.4 11% 8.5 33% PEO13228.1 hypothetical protein B4086_3570[Bacillus cereus] 40.8 40.8 11% 8.7 36% KLA04226.1 collagen-like protein[Bacillus thuringiensis] 40.8 40.8 10% 9.1 35% PFS10765.1 DUF4183domain-containing protein [Paenibacillus wynnii] 40.4 40.4 10% 9.3 26%WP_052088408.1 Gly-Xas-Xas repeat protein [Bacillus cereus] 41.2 41.215% 9.4 33% WP_061129112.1 hypothetical protein [Eimeria acervulina]41.6 41.6  8% 9.4 41% XP_013248017.1 hypothetical protein Tcan_05965[Toxocara canis] 40.4 40.4 10% 9.7 37% KHN81374.1 collagen-like protein[Bacillus wiedmannii] 40.4 40.4 10% 9.8 35% PEM96901.1 hypotheticalprotein CHLRE_02g089550v5 [Chlamydomonas reinhardtii] 41.2 41.2 10% 9.939% PNW86510.1 hypothetical protein REACYDRAFT_188388 [Fragilariopsiscylindrus 39.3 39.3  7% 9.9 37% OEU14454.1 CMP1102] hypothetical protein[Dactylosporangium aurantiacum] 41.2 41.2  9% 9.9 40% WP_03360279.1

TABLE 5A-1 Max Total Query E Description score score cover value IdentAccession

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

indicates data missing or illegible when filed

TABLE 5A-2 Max Total Query E Description score score cover value IdentAccession

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

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%

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%

.1

%

E−

%

.2

%

E−

%

.1

%

E−

%

.1

indicates data missing or illegible when filed

TABLE 5A-3 Max Total Query E Description score score cover value IdentAccession

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

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%

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%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

indicates data missing or illegible when filed

TABLE 5A-4 Max Total Query E Description score score cover value IdentAccession

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

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%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

indicates data missing or illegible when filed

TABLE 5A-5 Max Total Query E Description score score cover value IdentAccession

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

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%

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%

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%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

indicates data missing or illegible when filed

TABLE 5A-6 Max Total Query E Description score score cover value IdentAccession

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

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%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

indicates data missing or illegible when filed

TABLE 5A-7 Max Total Query E Description score score cover value IdentAccession

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

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E−

%

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%

E−

%

.1

%

E−

%

.1

indicates data missing or illegible when filed

TABLE 5A-8 Max Total Query E Description score score cover value IdentAccession

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

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E−

%

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%

E−

%

.1

%

E−

%

.1

indicates data missing or illegible when filed

TABLE 5A-9 Max Total Query E Description score score cover value IdentAccession

%

E−

%

.1

%

E−

%

.1

%

E−

%

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%

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%

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%

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%

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%

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%

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indicates data missing or illegible when filed

TABLE 5A-10 Max Total Query E Description score score cover value IdentAccession

%

E−

%

.1

%

E−

%

.1

%

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%

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%

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%

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%

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%

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%

.1

indicates data missing or illegible when filed

TABLE 5A-11 Max Total Query E Description score score cover value IdentAccession

%

E−

%

.1

%

E−

%

.1

%

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%

.1

%

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%

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%

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%

.1

indicates data missing or illegible when filed

TABLE 5A-12 Max Total Query E Description score score cover value IdentAccession

%

E−

%

.1

%

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%

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%

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%

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%

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indicates data missing or illegible when filed

TABLE 5A-13 Max Total Query E Description score score cover value IdentAccession

%

E−

%

.1

%

E−

%

.1

%

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%

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%

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%

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indicates data missing or illegible when filed

TABLE 5A-14 Max Total Query E Description score score cover value IdentAccession

%

E−

%

.1

%

E−

%

.1

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%

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%

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%

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%

.1

indicates data missing or illegible when filed

TABLE 5A-15 Max Total Query E Description score score cover value IdentAccession

%

E−

%

.1

%

E−

%

.1

%

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%

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%

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%

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%

.1

indicates data missing or illegible when filed

TABLE 5A-16 Max Total Query E Description score score cover value IdentAccession

%

E−

%

.1

%

E−

%

.1

%

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%

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%

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%

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%

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indicates data missing or illegible when filed

TABLE 5A-17 Max Total Query E Description score score cover value IdentAccession

%

E−

%

.1

%

E−

%

.1

%

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%

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%

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%

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%

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indicates data missing or illegible when filed

TABLE 5A-18 Max Total Query E Description score score cover value IdentAccession

%

E−

%

.1

%

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%

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%

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%

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%

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%

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indicates data missing or illegible when filed

TABLE 5A-19 Max Total Query E Description score score cover value IdentAccession

%

E−

%

.1

%

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%

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indicates data missing or illegible when filed

TABLE 5A-20 Max Total Query E Description score score cover value IdentAccession

%

E−

%

.1

%

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%

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%

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%

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%

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indicates data missing or illegible when filed

TABLE 5A-21 Max Total Query E Description score score cover value IdentAccession

%

E−

%

.1

%

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%

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indicates data missing or illegible when filed

TABLE 5A-22 Max Total Query E Description score score cover value IdentAccession

%

E−

%

.1

%

E−

%

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indicates data missing or illegible when filed

TABLE 5A-23 Max Total Query E Description score score cover value IdentAccession

%

E−

%

.1

%

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indicates data missing or illegible when filed

TABLE 5A-24 Max Total Query E Description score score cover value IdentAccession

%

E−

%

.1

%

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%

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%

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indicates data missing or illegible when filed

TABLE 5A-25 Max Total Query E Description score score cover value IdentAccession

%

E−

%

.1

%

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%

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%

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indicates data missing or illegible when filed

TABLE 5A-26 Max Total Query E Description score score cover value IdentAccession

%

E−

%

.1

%

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%

.1

%

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%

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indicates data missing or illegible when filed

TABLE 5A-27 Max Total Query E Description score score cover value IdentAccession

%

E−

%

.1

%

E−

%

.1

%

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indicates data missing or illegible when filed

TABLE 5A-28 Max Total Query E Description score score cover value IdentAccession

%

E−

%

.1

%

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%

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indicates data missing or illegible when filed

TABLE 5A-29 Max Total Query E Description score score cover value IdentAccession

%

E−

%

.1

%

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%

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indicates data missing or illegible when filed

TABLE 5A-30 Max Total Query E Description score score cover value IdentAccession

%

E−

%

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%

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.1

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E−

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%

E−

%

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%

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%

.1

indicates data missing or illegible when filed

TABLE 5A-31 Max Total Query E Description score score cover value IdentAccession

%

E−

%

.1

%

E−

%

.1

%

E−

%

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%

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%

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%

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%

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%

.1

indicates data missing or illegible when filed

TABLE 5A-32 Max Total Query E Description score score cover value IdentAccession

%

E−

%

.1

%

E−

%

.1

%

E−

%

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%

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%

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%

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%

.1

indicates data missing or illegible when filed

TABLE 5A-33 Max Total Query E Description score score cover value IdentAccession

%

E−

%

.1

%

E−

%

.1

%

E−

%

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%

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%

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%

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%

.1

indicates data missing or illegible when filed

TABLE 5A-34 Max Total Query E Description score score cover value IdentAccession

%

E−

%

.1

%

E−

%

.1

%

E−

%

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%

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%

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%

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%

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%

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indicates data missing or illegible when filed

TABLE 5A-35 Max Total Query E Description score score cover value IdentAccession

%

E−

%

.1

%

E−

%

.1

%

E−

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%

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%

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%

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indicates data missing or illegible when filed

TABLE 5A-36 Max Total Query E Description score score cover value IdentAccession

%

E−

%

.1

%

E−

%

.1

%

E−

%

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indicates data missing or illegible when filed

TABLE 5A-37 Max Total Query E Description score score cover value IdentAcession

%

E−

%

.1

%

E−

%

.1

%

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indicates data missing or illegible when filed

TABLE 5A-38 Max Total Query E Description score score cover value IdentAccession

%

E−

%

.1

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indicates data missing or illegible when filed

TABLE 5A-39 Max Total Query E Description score score cover value IdentAccession

%

%

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indicates data missing or illegible when filed

TABLE 5A-40 Max Total Query E Description score score cover value IdentAccession

%

%

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.1

%

%

.1

%

%

.1

indicates data missing or illegible when filed

TABLE 5A-41 Max Total Query E Description score score cover value IdentAccession

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

indicates data missing or illegible when filed

TABLE 5A-42 Max Total Query E Description score score cover value IdentAccession

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

indicates data missing or illegible when filed

TABLE 5A-43 Max Total Query E Description score score cover value IdentAccession

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

indicates data missing or illegible when filed

TABLE 5A-44 Max Total Query E Description score score cover value IdentAccession

%

%

.1

%

%

.1

%

%

.2

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

indicates data missing or illegible when filed

TABLE 5A-45 Max Total Query E Description score score cover value IdentAccession

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

indicates data missing or illegible when filed

TABLE 5A-46 Max Total Query E Description score score cover value IdentAccession

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

indicates data missing or illegible when filed

TABLE 5A-47 Max Total Query E Description score score cover value IdentAccession

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

indicates data missing or illegible when filed

TABLE 5A-48 Max Total Query E Description score score cover value IdentAccession

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

indicates data missing or illegible when filed

TABLE 5A-49 Max Total Query E Description score score cover value IdentAccession

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

indicates data missing or illegible when filed

TABLE 5A-50 Max Total Query E Description score score cover value IdentAccession

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

indicates data missing or illegible when filed

TABLE 5A-51 Max Total Query E Description score score cover value IdentAccession hypothetical protein [Oryza sativa Japonica Group] 39.3 39.322% 6.8 40% AAM93466.1 PREDICTED uncharacterized protein LOC9288486[Oryza sativa 39.3 39.3 22% 6.8 40% XP_015612992.1 Japonica Group]hypothetical protein BWK73_17640 [Thiothrix lacustris] 39.3 39.3 63% 7.229% OQX11438.1 KR domain-containing protein [Paenibacillus sp. Sc5Re-14]39.3 39.3 50% 7.2 29% WP_090739059.1 PREDICTED: LOW QUALITY PROTEIN:uncharacterized protein 39.3 39.3 27% 7.3 38% XP_008783575.1LOC103702788 [Phoenix dactylifera] SagB/ThcOx family dehydrogenase[Pseudomonas sp. M30-35] 39.3 39.3 21% 7.3 42% WP_087515666.1nitroreductase [Nitrosospira sp. NpAV] 39.3 39.3 90% 7.8 25% KIO49228.1SagB/ThcOx family dehydrogenase [Alistipes sp. ZOR0009] 38.5 38.5 62%7.7 27% WP_081981735.1 SagB/ThcOx family dehydrogenase [Nitrosospira sp.NpAV] 38.9 38.9 90% 7.8 25% WP_052494089.1 SagB/ThcOx familydehydrogenase [Bacillus jeotgali] 38.9 38.9 38% 7.8 33% WP_102262495.1nitroreductase family protein [Thermodesulfobacterium geofontis] 38.138.1 21% 7.8 39% PMP97705.1 MULTISPECIES: hypothetical protein[Streptomyces] 38.9 38.9 82% 8 28% WP_018105665.1 nitroreductase[Alcanivorax sp. DG881] 38.5 38.5 72% 8 27% WP_007151480.1 SagB/ThcOxfamily dehydrogenase [Azonexus hydrophilus] 38.9 38.9 80% 8.8 30%WP_028994401.1 SagB/ThcOx family dehydrogenase [Bacillus sp. RFGP41]38.9 38.9 59% 8.9 27% WP_106026268.1 SagB/ThcOx family dehydrogenase[Bacillus sp. FJAT-20673] 38.9 38.9 60% 9 27% WP_063575178.1MULTISPECIES: SagB/ThcOx family dehydrogenase [Bacillus] 38.9 38.9 59%9.1 27% WP_098187567.1 non-ribosomal peptide synthetase [Nodulariaspumigena] 38.9 38.9 89% 9.3 26% WP_006194152.1 SagB/ThcOx familydehydrogenase [Bacillus sp. FJAT-21352] 38.9 38.9 59% 9.4 27%WP_053536305.1 oxygen-insensitive NADPH nitroreductase 38.1 38.1 24% 9.728% WP_035342206.1 [Bacillus hemicellulosilylicus] SagB/ThcOx familydehydrogenase [Pseudomonas stutzeri] 38.9 38.9 26% 9.9 44%WP_102895655.1 SagB/ThcOx family dehydrogenase [Nocardiopsiskunsanensis] 38.9 38.9 61% 9.9 30% WP_017575210.1

TABLE 5B-1 Max Total Query E Description score score cover value IdentAccession

%

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

indicates data missing or illegible when filed

TABLE 5B-2 Max Total Query E Description score score cover value IdentAccession

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

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%

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%

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%

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%

.1

%

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%

.1

indicates data missing or illegible when filed

TABLE 5B-3 Max Total Query E Description score score cover value IdentAccession

%

E−

%

.1

%

E−

%

.1

%

E−

%

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%

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%

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%

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%

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%

.1

indicates data missing or illegible when filed

TABLE 5B-4 Max Total Query E Description score score cover value IdentAccession

%

E−

%

.1

%

E−

%

.1

%

E−

%

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%

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E−

%

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%

E−

%

.1

%

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%

.1

indicates data missing or illegible when filed

TABLE 5B-5 Max Total Query E Description score score cover value IdentAccession

%

E−

%

.1

%

E−

%

.1

%

E−

%

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%

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%

E−

%

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%

E−

%

.1

%

E−

%

.1

indicates data missing or illegible when filed

TABLE 5B-6 Max Total Query E Description score score cover value IdentAccession

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

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%

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E−

%

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%

E−

%

.1

%

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%

.1

indicates data missing or illegible when filed

TABLE 5B-7 Max Total Query E Description score score cover value IdentAccession

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

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%

E−

%

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%

E−

%

.1

%

E−

%

.1

indicates data missing or illegible when filed

TABLE 5B-8 Max Total Query E Description score score cover value IdentAccession

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

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%

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%

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%

.1

%

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%

.1

indicates data missing or illegible when filed

TABLE 5B-9 Max Total Query E Description score score cover value IdentAccession

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

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%

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%

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%

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%

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%

.1

indicates data missing or illegible when filed

TABLE 5B-10 Max Total Query E Description score score cover value IdentAccession

%

E−

%

.1

%

E−

%

.1

%

E−

%

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%

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%

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%

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%

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indicates data missing or illegible when filed

TABLE 5B-11 Max Total Query E Description score score cover value IdentAccession

%

E−

%

.1

%

E−

%

.1

%

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%

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%

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.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

indicates data missing or illegible when filed

TABLE 5B-12 Max Total Query E Description score score cover value IdentAccession

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

indicates data missing or illegible when filed

TABLE 5B-13 Max Total Query E Description score score cover value IdentAccession

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

indicates data missing or illegible when filed

TABLE 5B-14 Max Total Query E Description score score cover value IdentAccession

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

indicates data missing or illegible when filed

TABLE 5B-15 Max Total Query E Description score score cover value IdentAccession

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

indicates data missing or illegible when filed

TABLE 5B-16 Max Total Query E Description score score cover value IdentAccession

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

indicates data missing or illegible when filed

TABLE 5B-17 Max Total Query E Description score score cover value IdentAccession

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

indicates data missing or illegible when filed

TABLE 5B-18 Max Total Query E Description score score cover value IdentAccession

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

indicates data missing or illegible when filed

TABLE 5B-19 Max Total Query E Description score score cover value IdentAccession

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.3

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

indicates data missing or illegible when filed

TABLE 5B-20 Max Total Query E Description score score cover value IdentAccession

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

indicates data missing or illegible when filed

TABLE 5B-21 Max Total Query E Description score score cover value IdentAccession

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

E−

%

.1

indicates data missing or illegible when filed

TABLE 5B-22 Max Total Query E Description score score cover value IdentAccession

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

indicates data missing or illegible when filed

TABLE 5B-23 Max Total Query E Description score score cover value IdentAccession

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

indicates data missing or illegible when filed

TABLE 5B-24 Max Total Query E Description score score cover value IdentAccession

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

indicates data missing or illegible when filed

TABLE 5B-25 Max Total Query E Description score score cover value IdnetAccesssion

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.2

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

indicates data missing or illegible when filed

TABLE 5B-26 Max Total Query E Description score score cover value IdnetAccesssion

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

indicates data missing or illegible when filed

TABLE 5B-27 Max Total Query E Description score score cover value IdnetAccesssion

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

indicates data missing or illegible when filed

TABLE 5B-28 Max Total Query E Description score score cover value IdnetAccesssion

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

indicates data missing or illegible when filed

TABLE 5B-29 Max Total Query E Description score score cover value IdnetAccesssion

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

indicates data missing or illegible when filed

TABLE 5B-30 Max Total Query E Description score score cover value IdnetAccesssion

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

indicates data missing or illegible when filed

TABLE 5B-31 Max Total Query E Description score score cover value IdnetAccesssion

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

indicates data missing or illegible when filed

TABLE 5B-32 Max Total Query E Description score score cover value IdnetAccesssion

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.3

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

indicates data missing or illegible when filed

TABLE 6-1 Max Total Query E Description score score cover value IdnetAccesssion

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

indicates data missing or illegible when filed

TABLE 6-2 Max Total Query E Description score score cover value IdnetAccesssion

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

indicates data missing or illegible when filed

TABLE 6-3 Max Total Query E Description score score cover value IdnetAccesssion

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

indicates data missing or illegible when filed

TABLE 6-4 Max Total Query E Description score score cover value IdnetAccesssion

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

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%

.1

%

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%

.1

%

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%

.1

%

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%

.1

%

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%

.1

%

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%

.1

%

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%

.1

%

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%

.1

%

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%

.1

%

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%

.1

%

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%

.1

%

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%

.1

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%

.1

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%

.1

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%

.1

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%

.1

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%

.1

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%

.1

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%

.1

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%

.1

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%

.1

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%

.1

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%

.1

%

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%

.1

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%

.1

%

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%

.1

%

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%

.1

%

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%

.1

%

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%

.1

%

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%

.1

%

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%

.1

%

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%

.1

%

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%

.1

%

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%

.1

%

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%

.1

%

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%

.1

%

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%

.1

%

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%

.1

%

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%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

indicates data missing or illegible when filed

TABLE 6-5 Max Total Query E Description score score cover value IdnetAccesssion

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

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%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

indicates data missing or illegible when filed

TABLE 6-6 Max Total Query E Description score score cover value IdnetAccession

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

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%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

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%

.1

%

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%

.1

%

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%

.1

%

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%

.1

%

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%

.1

%

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%

.1

%

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%

.1

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%

.1

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%

.1

%

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%

.1

%

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%

.1

%

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%

.1

%

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%

.1

%

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%

.1

%

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%

.1

%

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%

.1

%

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%

.1

%

E−

%

.1

%

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%

.1

%

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%

.1

%

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%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

indicates data missing or illegible when filed

TABLE 6-7 Max Total Query E Description score score cover value IdnetAccession

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

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%

.1

%

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%

.1

%

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%

.1

%

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%

.1

%

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%

.1

%

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%

.1

%

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%

.1

%

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%

.1

%

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%

.1

%

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%

.1

%

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%

.1

%

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%

.1

%

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%

.1

%

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%

.1

%

E−

%

.1

%

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%

.1

%

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%

.1

%

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%

.1

%

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%

.1

%

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%

.1

%

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%

.1

%

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%

.1

%

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%

.1

%

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%

.1

%

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%

.1

%

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%

.1

%

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%

.1

%

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%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

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%

.1

%

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%

.1

%

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%

.1

%

E−

%

.1

%

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%

.1

%

E−

%

.1

%

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%

.1

%

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%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

indicates data missing or illegible when filed

TABLE 6-8 Max Total Query E Description score score cover value IdnetAccession

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

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%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

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%

.1

%

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%

.1

%

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%

.1

%

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%

.1

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%

.1

%

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%

.1

%

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%

.1

%

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%

.1

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%

.1

%

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%

.1

%

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%

.1

%

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%

.1

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%

.1

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%

.1

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%

.1

%

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%

.1

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%

.1

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%

.1

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%

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%

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%

.1

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%

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%

.1

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%

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%

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%

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%

.1

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%

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%

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%

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%

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%

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%

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%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

indicates data missing or illegible when filed

TABLE 6-9 Max Total Query E Description score score cover value IdnetAccession

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

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%

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%

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%

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%

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%

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%

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%

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%

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%

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%

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%

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%

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%

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%

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%

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%

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%

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%

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%

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%

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%

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%

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%

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%

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%

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%

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%

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%

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%

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%

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%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

indicates data missing or illegible when filed

TABLE 6-10 Max Total Query E Description score score cover value IdnetAccession

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

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%

.1

%

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%

.1

%

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%

.1

%

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%

.1

%

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%

.1

%

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%

.1

%

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%

.1

%

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%

.1

%

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%

.1

%

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%

.1

%

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%

.1

%

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%

.1

%

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%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

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%

.1

%

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%

.1

%

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%

.1

%

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%

.1

%

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%

.1

%

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%

.1

%

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%

.1

%

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%

.1

%

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%

.1

%

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%

.1

%

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%

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%

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%

.1

%

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%

.1

%

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%

.1

%

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%

.1

%

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%

.1

%

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%

.1

%

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%

.1

%

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%

.1

%

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%

.1

%

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%

.1

%

E−

%

.1

%

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%

.1

%

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%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

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%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

indicates data missing or illegible when filed

TABLE 6-11 Max Total Query E Description score score cover value IdnetAccession

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

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%

.1

%

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%

.1

%

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%

.1

%

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%

.1

%

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%

.1

%

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%

.1

%

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%

.1

%

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%

.1

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%

.1

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%

.1

%

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%

.1

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%

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%

.1

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%

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.1

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%

.1

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%

.1

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%

.1

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%

.1

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%

.1

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%

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.1

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%

.1

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%

.1

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%

.1

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%

.1

%

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%

.1

%

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%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

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%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

indicates data missing or illegible when filed

TABLE 6-12 Max Total Query E Description score score cover value IdnetAccession

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

%

.1

indicates data missing or illegible when filed

Aable 6-13 Max Total Query E Description score score cover value IdentAccession hypothetical protein [Kutzneria sp. 744] 45.4 45.4 96% 0.5526% WP_084579540.1 lantibiotic dehydratase family protein [Frankia sp.Allo2] 45.1 45.1 12% 0.72 38% KFB02770.1 hypothetical protein[Streptomyces venezuelae] 44.7 44.7 49% 0.8 28% WP_055642955.1hypothetical protein [Streptomyces rubidus] 44.3 44.3 12% 1.1 35%WP_069466882.1 lantibiotic dehydratase [Streptacidiphilus anmyonensis]43.9 43.9 13% 1.3 34% WP_042429246.1 hypothetical protein [Streptomycesmegasporus] 42.7 42.7 16% 3.1 31% WP_031510724.1 hypothetical protein[Streptomyces sp. NRRL S-475] 42.7 42.7 13% 3.3 33% WP_030849796.1MULTISPECIES: hypothetical protein [Rhodococcus] 42.7 42.7 11% 3.7 36%WP_051730664.1 hypothetical protein [Kitasatospora phosalacinea] 42.742.7 33% 3.7 35% WP_033255123.1 hypothetical protein [Streptomyces sp.NRRL S-146] 42.4 42.4 13% 4.1 33% WP_037821239.1 hypothetical protein[Streptomyces sp. S10(2016)] 42.4 42.4 13% 4.2 33% WP_062927424.1hypothetical protein [Streptomyces kanamyceticus] 42.4 42.4 11% 4.4 36%WP_079043830.1 dehydratase [Nonomuraea sp. WU8817] 42.4 42.4 27% 4.7 31%ACS83767.1 hypothetical protein [Streptomyces flacochromogenes] 42.442.4 16% 4.9 28% WP_030320403.1 lantibiotic dehydratasedomain-containing protein 42 42 41% 8.4 28% AJC59437.1 [Streptomyces sp.769] hypothetical protein [Streptomyces sp. RSD-27] 41.6 41.6 11% 7.437% KIF07651.1 hypothetical protein [Streptomyces sp. Ag82_G6-1] 41.641.6 13% 7.5 33% WP_097217204.1 hypothetical protein [Micromonosporaeburnea] 41.6 41.6 15% 8.2 31% WP_091121031.1 beta-galactosidase[Streptomyces laurenti] 41.2 41.2 11% 9.7 36% BAU87544.1 hypotheticalprotein [Actinomadura oligospora] 41.2 41.2 31% 10 30% WP_026413556.1

TABLE 7-1 Max Total Query E Description score score cover value IdnetAccession

%

%

.1

%

%

.1

%

%

.1

%

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

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%

.1

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%

.1

%

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%

.1

%

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%

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%

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%

.1

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%

.1

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%

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%

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%

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%

.1

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%

.1

%

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%

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%

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%

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%

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%

.1

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%

.1

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%

.1

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%

.1

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%

.1

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%

.1

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%

.1

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%

.1

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%

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%

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%

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%

.1

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%

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%

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%

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%

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%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

indicates data missing or illegible when filed

TABLE 7-2 Max Total Query E Description score score cover value IdnetAccession

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

E−

%

.1

%

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%

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%

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%

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%

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%

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%

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%

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%

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%

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%

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%

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%

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%

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%

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%

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%

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%

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%

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%

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%

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%

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%

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%

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%

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%

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%

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%

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%

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%

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%

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%

.1

%

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%

.1

indicates data missing or illegible when filed

TABLE 7-3 Max Total Query E Description score score cover value IdnetAccession

%

E−

%

.1

%

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%

.1

%

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%

.1

%

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%

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%

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%

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%

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%

.1

indicates data missing or illegible when filed

TABLE 7-4 Max Total Query E Description score score cover value IdnetAccession

%

E−

%

.1

%

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%

.1

%

E−

%

.1

%

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%

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indicates data missing or illegible when filed

TABLE 7-5 Max Total Query E Description score score cover value IdnetAccession

%

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%

.1

%

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.1

%

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%

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indicates data missing or illegible when filed

TABLE 7-6 Max Total Query E Description score score cover value IdnetAccession hypothetical protein [Thermobispora bispora] 41.2 41.2 82% 2.726% WP_050760415.1 Chain A, Pyridine synthase, TbtD, from thiomuracinbiosynthesis 41.2 41.2 82% 2.7 26% 5WA3_A hypothetical proteinAQJ66_16295 [Streptomyces bungoensis] 41.2 41.2 88% 2.8 30% KUN84096.1unknown [Streptomyces hygroscopious] 40.8 40.8 86% 3.5 28% ACS50127.1lantibiotic biosynthesis protein [Fulvivirga imtechensis] 40.8 40.8 19%3.7 35% WP_009582377.1 hypothetical protein [Streptomyces lydicus] 40.840.8 84% 3.9 25% WP_069569928.1 hypothetical protein [Nonomuraeafuscirosea] 40.8 40.8 95% 3.9 26% WP_106246561.1 lantibioticbiosynthesis protein [Streptomyces laurentii] 40.4 40.4 22% 4.7 33%BAU81447.1 lantibiotic biosynthesis protein [Thermobifida halotolerans]40.4 40.4 31% 4.9 31% WP_084012656.1 serline-tRNA ligase [CandidatusStaskawiczbaterira bacterium 40.4 40.4 22% 5.3 27% OGZ66026.1RIFCSPHIGHO2_01_FULL_41_41] hypothetical protein [Streptomycesangustmycelicus] 40 40 84% 7.7 28% WP_086715354.1 lantibioticbiosynthesis protein [Streptomyces pathocidin] 39.7 39.7 32% 7.8 31%WP_055471434.1 hypothetical protein [Herbidospora cretacea] 39.7 39.721% 8.4 36% WP_030455318.1 MULTISPECIES: SDR family NAD(P)-dependentoxidoreductase 39.3 39.3 15% 8.8 42% WP_027506043.1 [Rhoducoccus]hypothetical protein [Herbidospora mongoliensis] 39.3 39.3 21% 9.1 36%WP_066361444.1 MULTISPECIES: hypothetical protein [Nonomuraea] 39.7 39.731% 9.4 32% WP_082534571.1 Lantibiotic biosynthesis dehydratase C-term[Nonomuraea pusilia] 39.7 39.7 31% 9.7 32% SEL93819.1

The reaction of the α,β-unsaturated amino acid-forming enzyme with apeptide according to the present invention is performed in the presenceof a cosubstrate for glutamylation reaction and aminoacylation enzyme.In this context, the cosubstrate for glutamylation reaction and theaminoacylation enzyme may be derived from a cell-free translation systemor may be other than those contained in the cell-free translationsystem. Specifically, the reaction of the α,β-unsaturated aminoacid-forming enzyme with a peptide according to the present inventionmay be performed using a cosubstrate for glutamylation reaction andaminoacylation enzyme in a cell-free translation system when thecell-free translation system contains them, and may be performed in thepresence of a cosubstrate for glutamylation reaction and/oraminoacylation enzyme that is not contained in the cell-free translationsystem, by separately adding them. Whether to use a cosubstrate andaminoacylation enzyme contained in a cell-free translation system or touse a cosubstrate and aminoacylation enzyme other than the cell-freetranslation system can be appropriately selected according to the typeof the α,β-unsaturated amino acid-forming enzyme used, etc.

From homology information on amino acid sequences by HHpred, LazB hasexhibited 13% identity to an N-terminal region of biosynthetic enzymeNisB for class I lanthipeptide Nisin carried by Lactococcus lactis. NisBcatalyzes the formation of an α,β-unsaturated amino acid such asdehydroalanine or dehydrobutyrine by the dehydration of Ser or Thr inNisin biosynthesis.

For RiPPs compounds, it has been reported that the α,β-unsaturated aminoacid such as dehydroalanine or dehydrobutyrine is formed through thedehydration reaction of Ser or Thr. The Ser or Thr dehydration mechanismis reportedly divided broadly into two different mechanisms. One of themis a tRNA-dependent dehydration mechanism seen in thiopeptides or classI lanthipeptides, and the other one is an ATP-dependent dehydrationmechanism seen in class II to IV lanthipeptides.

The tRNA-type dehydration mechanism has been revealed for NisB. As shownbelow, a dehydroamino acid is formed by glutamylation which adds aglutamic acid aminoacylated to glutamyl-tRNA to a hydroxy group, andelimination of the glutamic acid.

It is known that the α,β-unsaturated amino acid formation of otherthiopeptides is based on the tRNA-type dehydration mechanism (NatureVolume 517, pages 509-512, (2015)). Since LazB has high homology with anN-terminal region of NisB, LazB presumably catalyzes glutamylationreaction using glutamyl-tRNA^(Glu).

Thus, in the present invention, glutamyl-tRNA^(Glu) is used as acosubstrate for reaction in the formation of an α,β-unsaturated aminoacid. In the present invention, particularly, when LazB and LazF, and/orenzymes having homology with them are used as the α,β-unsaturated aminoacid-forming enzyme, tRNA^(Glu) is not particularly limited as long asthe tRNA^(Glu) is at least accepted by the α,β-unsaturated aminoacid-forming enzyme. From the viewpoint that the reaction proceedsefficiently, it is preferred to use actinomycete-derived tRNA^(Glu)and/or E. coli-derived tRNA^(Glu), and it is more preferred to usetRNA^(Glu) derived from an actinomycete Streptomyces lactacystinaeuswhich is a lactazole-producing microbe and/or tRNA^(Glu) derived fromStreptomyces lividans which is used as a host heterologously expressinglactazole.

Also, aminoacylation enzyme GluRS is used for formingglutamyl-tRNA^(Glu) from tRNA^(Glu). Particularly, when LazB and LazF,and/or enzymes having homology with them are used as the α,β-unsaturatedamino acid-forming enzyme, GluRS is not particularly limited as long asthe tRNA^(Glu) used is glutamylated. Actinomycete-derived GluRS and/orE. coli-derived GluRS is preferred, and GluRS derived from anactinomycete Streptomyces lactacystinaeus which is a lactazole-producingmicrobe and/or GluRS derived from Streptomyces lividans which is used asa host heterologously expressing lactazole is more preferred. Theactinomycete-derived GluRS may be GluRS extracted from the actinomyceteor may be obtained as a recombinant protein of GluRS within theactinomycete genome. The E. coli-derived GluRS may be GluRS extractedfrom E. coli or may be obtained as a recombinant protein of GluRS fromthe E. coli genome.

The peptide represented by the formula (II) may be prepared, forexample, from a precursor peptide represented by the formula (III) bythe action of the azole ring-forming enzyme, the α,β-unsaturated aminoacid-forming enzyme and the macrocyclase, or may be prepared by anorganic chemical approach known in the art with a precursor peptiderepresented by the formula (III) as a starting material. Alternatively,the peptide represented by the formula (II) may be prepared inaccordance with a peptide synthesis method known in the art, forexample, an organic chemical approach described in J. Am. Chem. Soc2015, 137, 3494-3497, and J. Am. Chem. Soc. 2016, 138, 13461-13464.

Specifically, the peptide represented by the formula (II) can beobtained, for example, by allowing a general acid or base catalyst toact on the precursor peptide represented by the formula (III) so that acarbonyl group and a hydroxy group or a thiol group in the peptide areintramolecularly dehydrated and condensed to form an azole ring, thenconverting a hydroxy group in the peptide to a leaving group, andallowing a general base to act on the resultant to form anα,β-unsaturated amino acid. Alternatively, the precursor peptiderepresented by the formula (III) may be prepared using amino acidshaving a selenium atom at position γ, such as selenocysteine,phenylselenocysteine and selenolysine, and induced into selenoxide bytreatment with an oxidizing agent such as hydrogen peroxide, followed byβ elimination to form an α,β-unsaturated amino acid.

In the present specification, the “cell-free translation system” refersto a translation system involving no cell. For example, E. coliextracts, wheat germ extracts, rabbit erythrocyte extracts or insectcell extracts can be used as the cell-free translation system.Alternatively, a reconstituted cell-free translation system may be used,which is constructed by reconstituting a ribosomal protein,aminoacyl-tRNA synthetase (aaRS), ribosomal RNA, amino acid, rRNA, GTP,ATP, translation initiation factor (IF) elongation factor (EF), releasefactor (RF) and ribosome recycling factor (RRF), respectively purified,and other factors necessary for translation.

The system may contain RNA polymerase in order to also performtranscription from DNA. A commercially available cell-free translationsystem can be used, for example, RTS-100(registered trademark) fromRoche Diagnostics K.K. as an E. coli-derived system,PURESYSTEM(registered trademark) from Post Genome Institute (PGI) andPURExpress® In Vitro Protein Synthesis Kit from New England BioLabs Inc.as a reconstituted translation system, and a system using wheat germextracts from ZOEGENE Corp. or CellFree Sciences Co., Ltd.

For example, techniques described in the following documents are knownin the art as systems using E. coli ribosome: H. F. Kung et al., 1977.The Journal of Biological Chemistry Vol. 252, No. 19, 6889-6894; M. C.Gonza et al., 1985, Proceeding of National Academy of Sciences of theUnited States of America Vol. 82, 1648-1652; M. Y. Pavlov and M.Ehrenberg, 1996, Archives of Biochemistry and Biophysics Vol. 328, No.1, 9-16; Y. Shimizu et al., 2001, Nature Biotechnology Vol. 19, No. 8,751-755; H. Ohashi et al., 2007, Biochemical and Biophysical ResearchCommunications Vol. 352, No. 1, 270-276.

According to the cell-free translation system, an expression product canbe obtained with high purity without being purified.

The cell-free translation system of the present invention may besupplemented with factors necessary for transcription and thereby usednot only in translation but in transcription.

One aspect of the present invention is a compound library containing twoor more cyclic compounds represented by the formula (I), wherein atleast one of the cyclic compounds represented by the formula (I) is in anon-natural form.

The non-natural form refers to a form structurally differing fromnaturally occurring lactazole or the like.

One aspect of the present invention is a screening method foridentifying a cyclic compound represented by the formula (I) which bindsto a target substance, including: a step of contacting a compoundlibrary produced by the production method of the present invention orthe compound library of the present invention with the target substance,followed by incubation; and a step of selecting a cyclic compoundrepresented by the formula (I) bound with the target substance.

One aspect of the present invention is a screening kit for identifying acyclic compound represented by the formula (I) which binds to a targetsubstance, containing a compound library produced by the productionmethod of the present invention or the compound library of the presentinvention.

One aspect of the present invention is a method for producing a cycliccompound represented by the formula (I), including a step of allowing amacrocyclase in vitro to act on a peptide represented by the formula(II) and forming the nitrogen-containing 6-membered ring A whileeliminating LP to form the cyclic compound represented by the formula(I).

One aspect of the present invention is a method for producing a cycliccompound represented by the formula (I), including: a step of producingmRNA encoding a precursor peptide represented by the formula (III); astep of expressing the precursor peptide by a cell-free translationsystem using the mRNA to produce a first peptide; a step of reacting anazole ring-forming enzyme with the first peptide, so that an azole ringis formed on at least Y′(11) in the precursor peptide to produce asecond peptide; a step of reacting an α,β-unsaturated amino acid-formingenzyme with the second peptide in the presence of cosubstrate tRNA^(Glu)for glutamylation reaction and aminoacylation enzyme GluRS, so that atleast X′ and Y′(12) in the precursor peptide are converted toα,β-unsaturated amino acid residues to produce a third peptide; and astep of reacting a macrocyclase with the third peptide and forming anitrogen-containing 6-membered ring while eliminating LP to form acyclic compound represented by the formula (I).

When the method for producing a compound library and the method forproducing a cyclic compound according to the present invention includesteps of producing a first peptide library or a first peptide, producinga second peptide library or a second peptide, producing a third peptidelibrary or a third peptide and producing a cyclic compound, these stepsalso encompass an embodiment in which the reactions are performed atonce by adding enzymes or cosubstrates to one system, i.e., anembodiment in which these production steps proceed in a series ofreactions in the system, and also encompass an embodiment in which thereactions are performed stepwise by adding and reacting enzymes inorder.

Specific embodiments encompassed by the method for producing a compoundlibrary and the method for producing a cyclic compound according to thepresent invention include:

an embodiment which includes first adding an azole ring-forming enzymeto a system containing precursor peptides or a puromycin-bound mRNAlibrary, followed by reaction, next adding an α,β-unsaturated aminoacid-forming enzyme, optionally with cosubstrate tRNA^(Glu) forglutamylation reaction and/or aminoacylation enzyme GluRS, followed byreaction, and further adding a macrocyclase, followed by reaction(pattern I);

an embodiment which includes first adding cosubstrate tRNA^(Glu) forglutamylation reaction and/or aminoacylation enzyme GluRS, if necessary,to a system containing precursor peptides or a puromycin-bound mRNAlibrary, adding an azole ring-forming enzyme and an α,β-unsaturatedamino acid-forming enzyme, followed by reaction, and next adding amacrocyclase, followed by reaction (pattern II);

an embodiment which includes first adding an azole ring-forming enzymeto a system containing precursor peptides or a puromycin-bound mRNAlibrary, followed by reaction, next adding cosubstrate tRNA^(Glu) forglutamylation reaction and/or aminoacylation enzyme GluRS, if necessary,adding an α,β-unsaturated amino acid-forming enzyme and macrocyclase,followed by reaction (pattern III); and

an embodiment which includes first adding cosubstrate tRNA^(Glu) forglutamylation reaction and/or aminoacylation enzyme GluRS, if necessary,to a system containing precursor peptides or a puromycin-bound mRNAlibrary, and adding an azole ring-forming enzyme, an α,β-unsaturatedamino acid-forming enzyme and a macrocyclase, followed by reaction(pattern IV).

EXAMPLES

<Synthesis of Precursor Peptide>

In order to reconstruct lactazole biosynthesis in vitro, a precursorpeptide was prepared using a cell-free translation system. It isconsidered that C-terminal several amino acids of LazA are cleaved bycellular endogenous protease. Accordingly, LazA* tailored to a purifiedproduct of lactazole A was synthesized by truncating C-terminal threeresidues (QDM). The sequences of LazA and LazA* are shown in FIG. 1 .

(Preparation of DNA Template)

In order to synthesize the precursor peptide using a cell-freetranslation system, a DNA template was first prepared.

An attempt was made to prepare a DNA template containing a nucleotidesequence encoding the precursor peptide LazA as well as a T7 promotersequence, a SD sequence and a ribosomal binding sequence. For thepromoter sequence, the SD sequence and the ribosomal binding sequence, aDNA template used in a FIT-GS system (goadsporin) was referred to(Ozaki, T. et al. Dissection of goadsporin biosynthesis by in vitroreconstitution leading to designer analogues expressed in vivo. NatCommun 8, 14207, doi:10.1038/ncomms14207 (2017)). FIG. 2 shows aschematic view of the lazA DNA template.

The nucleotide sequence encoding the precursor peptide needed to bechanged from actinomycete-type codons into E. coli type so that thecell-free translation system can utilize E. coli-derived RNA polymeraseor ribosome. Hence, the DNA template was designed so as to optimize thenucleotide sequence on the basis of the usage frequency of E. coli codonas shown in Table 8. The designed nucleotide sequence encoding theprecursor peptide is shown in FIG. 3 .

TABLE 8 Usage frequency of E. coli codon Order of precedence 1 2 3 4 5Ala GCA GCT GCG Cys TGT TGC Asp GAC GAT Glu GAA GAG Phe TTT TTC Gly GGTGGC GGG His CAT CAC Ile ATT ATC Lys AAG AAA Leu CTG TTA CTT TTG Met ATGAsn AAT AAC Pro CCG CCA CCT Gln CAA CAG Arg CGT CGC Ser TCT TCA AGC AGTThr ACC ACT ACG ACA Val GTT GTG GTA Trp TGG Tyr TAT TAC

Subsequently, the preparation of the designed DNA template wasattempted. Since the nucleotide sequence of the DNA template was 214base pairs, the DNA template was prepared by primer extension and PCRusing six primers. FIG. 4 shows a schematic view of the primerextension. Tables 9 to 15 show the reaction composition and reactionconditions of the primer extension system, and the reaction compositionand reaction conditions of the PCR system.

The prepared DNA template was confirmed by electrophoresis to have basepairs having the length of interest. The DNA template having theconfirmed length was purified using FastGene/PCR Extraction Kit (NipponGenetics Co., Ltd.) and eluted with 20 μl of MilliQ water.

TABLE 9 Primer extension reaction composition KOD-Plus-neo (Toyobo Co.,Ltd.) 2.0 (μl) 10 × KOD-Plus-neo Buffer 10 2 mM dNTPs 10 25 mM MgSO₄ 6.050 μM F1 primer 2.0 50 μM R1 primer 2.0 MilliQ water 68 Total 100 μl

TABLE 10 Primer extension reaction conditions 94° C. 1.0 min 50° C. 1.0min {close oversize bracket} 5 cycles 68° C. 1.0 min

TABLE 11 1st PCR reaction composition KOD-Plus-neo 2.0 (μl) 10 ×KOD-Plus-neo Buffer 10 2 mM dNTPs 10 25 mM MgSO₄ 6.0 50 μM F2 primer 1.050 μM R2 primer 1.0 MilliQ water 65 Extension product 5.0 Total 100 μl

TABLE 12 2nd PCR reaction composition KOD-Plus-neo 2.0 (μl) 10 ×KOD-Plus-neo Buffer 10 2 mM dNTPs 10 25 mM MgSO₄ 6.0 50 μM F2 primer 1.050 μM R3 primer 1.0 MilliQ water 69.5 1st PCR product 0.5 Total 100 μl

TABLE 13 3rd PCR reaction composition KOD-Plus-neo 2.0 (μl) 10 ×KOD-Plus-neo Buffer 10 2 mM dNTPs 10 25 mM MgSO₄ 6.0 50 μM F2 primer 1.050 μM R4 primer 1.0 MilliQ water 69.5 2nd PCR product 0.5 Total 100 μl

TABLE 14 1st PCR and 2nd PCR reaction conditions 94° C. 40 min 50° C. 40min {close oversize bracket} 5 cycles 68° C. 40 min

TABLE 15 3rd PCR reaction conditions 94° C. 40 min 50° C. 40 min {closeoversize bracket} 14 cycles 68° C. 40 min

(Translation Reaction in Cell-Free Translation System)

Next, translation reaction was performed in a cell-free translationsystem with the DNA template prepared in the preceding section(Preparation of DNA template) to prepare precursor peptide LazA.

The cell-free translation system used in this experiment was the oneprepared by the Suga Laboratory of Department of Chemistry, School ofScience, The University of Tokyo. The preparation of the cell-freetranslation system is described in Goto, Y., Katoh, T. & Suga, H.Flexizymes for genetic code reprogramming. Nat Protoc 6, 779-790,(2011). E. coli A19-derived ribosome, translation elongation factors(IF1, IF2, and IF3), translation elongation factors (EF-G, EF-Tu, andEF-Ts), translation release factors (RF2, RF3, and RRF), aminoacyl-tRNAsynthetase, methionyl-tRNA formyltransferase, T7 RNA polymerase,creatine kinase, myokinase, inorganic pyrophosphatase and nucleosidediphosphate kinase were used as constituent proteins of the cell-freetranslation system.

The cell-free translation system employed two divided solutions,Solution A containing cofactors, etc. and Solution B containingproteins, preserved at −80° C. The composition of the Solution A and theSolution B used is as described in Nature Protocols, 2011. TheN-terminal amino acid of the peptide to be synthesized becomesformylmethionine by the action of the methionyl-tRNA formyltransferase.

In vitro translation reaction was performed using the cell-freetranslation system with the prepared DNA template. The composition ofthe in vitro translation system is shown in Table 16. The reaction wasperformed by incubation at 37° C. for 30 minutes using a vapor-phaseincubator.

After the in vitro translation reaction, desalting operation wasperformed using zip tip C18. Subsequently, the desalted translatedpeptide was analyzed by MALDI-TOFMS using sinapic acid as a matrix. As aresult of the analysis by MALDI-TOF MS, a peak was observed at [M+H]⁺5672.4 (calculated value is [M+H]⁺ 5672.6), confirming that theprecursor peptide LazA* of interest was able to be synthesized.

TABLE 16 Reaction conditions Solution A version 7S 0.273 μl Solution Bversion10.1 0.320 μl DNA template 0.25 μl 5 mM mixed solution of 20amino acids 0.25 μl MilliQ 1.407 μl Total 2.5 μl

<Preparation of LazB, LazC, LazD, LazE, LazF, Cosubstrate and GluRS>

The enzymes used in the production of libraries were prepared asfollows.

(Preparation of LazB)

lazB-opt/pET26b was transferred to E. coli BL21 (DE3) for proteinexpression by heat shock method to obtain transformants. One colonythereof was precultured at 37° C. for 16 hours using 6 ml of LB medium.4 ml of the resulting cultures was inoculated to 200 ml of ZYM-5052medium. Subsequently, main culture was performed at 180 rpm at 18° C.for 20 hours. The bacterial cells thus cultured were centrifuged at4720×g for 10 minutes for harvest and then stored at −80° C. For theextraction of the recombinant protein, the bacterial cells weresuspended in 50 mM Tris-HCl pH 8.0, 500 mM NaCl and 10 mM imidazole pH8.0. The suspended bacterial cells were ultrasonically disrupted, andthe suspension was centrifuged at 10300×g at 4° C. for 30 minutes. Fromthe obtained supernatant, the protein was subjected toHis-tag-purification using Bio-Scale™ Mini Profinity™ IMAC cartridge(Bio-Rad Laboratories, Inc.). The cartridge was washed with the samebuffer as used in the suspension, followed by elution with a bufferhaving an imidazole concentration elevated to 200 mM. The elutionfraction was desalted with Bio-Gel P-6 desalting cartridge (Bio-RadLaboratories, Inc.). The buffer was replaced with 25 mM HEPES pH 8.0,500 mM NaCl and 5% glycerol. The LazB purification fraction wassubjected to polyacrylamide gel electrophoresis (SDS-PAGE). As a result,a band corresponding to His-Tag-bound LazB was observed at a molecularweight of approximately 96 kDa, confirming that the protein of interestwas correctly obtained. The concentration calculated from absorbance atUV 280 nm was 17.9 μM (approximately 1.7 mg/ml).

(Preparation of LazC)

lazC-opt/pET26b was transferred to E. coli BL21 (DE3) for proteinexpression by heat shock method to obtain transformants. One colonythereof was precultured at 37° C. for 16 hours using 6 ml of LB medium.4 ml of the resulting cultures was inoculated to 200 ml of ZYM-5052medium. Subsequently, main culture was performed at 180 rpm at 18° C.for 20 hours. The bacterial cells thus cultured were purified anddesalted in the same manner as in LazB. The buffer was replaced with 25mM HEPES pH 6.8 and 500 mM NaCl. The LazC purification fraction wassubjected to SDS-PAGE. As a result, a band corresponding toHis-Tag-bound LazC was observed at a molecular weight of approximately45 kDa, confirming that the protein of interest was correctly obtained.The concentration calculated from absorbance at UV 280 nm was 23.0 μM(approximately 1.0 mg/ml).

(Preparation of LazD)

Although coexpression with pET26b vector, pET16 vector and chaperon,etc. was attempted for the purification of LazD, no solubilized proteinwas obtained in any of the cases due to low expression levels andinsolubilization. Accordingly, pCold vector of a cold-shock expressionsystem was attempted.

lazD-opt/pColdII was transferred to E. coli BL21 (DE3) for proteinexpression by heat shock method to obtain transformants. One colonythereof was precultured at 37° C. for 16 hours using 20 ml of LB medium.16 ml of the resulting cultures was inoculated to 800 ml of LB medium.Subsequently, the bacterial cells were cultured at 150 rpm at 37° C. for2 hours and then cooled in ice. IPTG was added thereto with a finalconcentration of 0.1 mM, and the bacterial cells were cultured at 180rpm at 15° C. for 18 hours. The bacterial cells thus cultured werecentrifuged at 4720×g for 10 minutes for harvest and then immediatelysuspended in 50 mM Tris-HCl pH 8.0, 500 mM NaCl, 10 mM imidazole pH 8.0and 2 mM DTT. The suspended bacterial cells were ultrasonicallydisrupted, and the suspension was centrifuged at 10300×g at 4° C. for 30minutes. From the obtained supernatant, the protein was subjected toHis-tag-purification using Bio-Scale™ Mini Profinity™ IMAC cartridge.The cartridge was washed with the same buffer as used in the suspension,followed by elution with a buffer having an imidazole concentrationelevated to 200 mM. The elution fraction was desalted with Bio-Gel P-6desalting cartridge. The buffer was replaced with 25 mM HEPES pH 8.0,500 mM NaCl and 2 mM DTT. The LazD purification fraction was subjectedto SDS-PAGE. As a result, a band corresponding to His-Tag-bound LazD wasobserved at a molecular weight of approximately 58 kDa, confirming thatthe protein of interest was correctly obtained. The fraction wasconcentrated approximately 4-fold by ultrafiltration using a 30 kDaultrafiltration membrane. The concentration of the concentrated fractioncalculated from absorbance at UV 280 nm was 41.7 μM (approximately 2.4mg/ml).

(Preparation of LazE)

Although coexpression with pET26b vector, pET16 vector and chaperon,etc. was attempted for the expression and purification of LazE, as withLazD, no solubilized protein was obtained in any of the cases due to lowexpression levels and insolubilization. Accordingly, the pCold vector ofa cold-shock expression system was attempted.

lazE-opt/pColdII was transferred to E. coli BL21 (DE3) for proteinexpression by heat shock method to obtain transformants. One colonythereof was precultured at 37° C. for 16 hours using 20 ml of LB medium.16 ml of the resulting cultures was inoculated to 800 ml of LB medium.Subsequently, the bacterial cells were cultured at 150 rpm at 37° C. for2 hours and then cooled in ice. IPTG was added thereto with a finalconcentration of 0.1 mM, and the bacterial cells were cultured at 180rpm at 15° C. for 18 hours. The bacterial cells thus cultured werepurified and desalted in the same manner as in LazD. The LazEpurification fraction was subjected to SDS-PAGE. As a result, a bandcorresponding to His-Tag-bound LazE was observed at a molecular weightof approximately 74 kDa, confirming that the protein of interest wascorrectly obtained. The fraction was concentrated approximately 7-foldby ultrafiltration using a 30 kDa ultrafiltration membrane. Theconcentration of the concentrated fraction calculated from absorbance atUV 280 nm was 22.6 μM (approximately 1.7 mg/ml).

(Preparation of LazF)

lazF-opt/pET26b was transferred to E. coli BL21 (DE3) for proteinexpression by heat shock method to obtain transformants. One colonythereof was precultured at 37° C. for 16 hours using 6 ml of LB medium.4 ml of the resulting cultures was inoculated to 200 ml of ZYM-5052medium. Subsequently, main culture was performed at 180 rpm at 18° C.for 20 hours. The bacterial cells thus cultured were purified anddesalted in the same manner as in LazB. The buffer was replaced with 25mM HEPES pH 6.8 and 500 mM NaCl. The LazF purification fraction wassubjected to SDS-PAGE. As a result, a band corresponding toHis-Tag-bound LazF was observed at a molecular weight of approximately61 kDa, confirming that the protein of interest was correctly obtained.The purified LazF assumed yellow color which indicated that LazF wasbound with flavin. Yamashita et al. have revealed that FMN isnoncovalently bound with LazF (Ozaki, T. et al. Dissection of goadsporinbiosynthesis by in vitro reconstitution leading to designer analoguesexpressed in vivo. Nat Commun 8, 14207, (2017)). The concentration ofLazF calculated by the Bradford method was 32.9 μM (approximately 2.0mg/ml).

(Preparation of Cosubstrate)

tRNA^(Glu) derived from Lactazole-producing microbe, which serves as acosubstrate for glutamylation reaction mediated by LazB was prepared byin vitro transcription.

(1) Synthesis of DNA Template

First, a tRNA^(Glu) sequence was found from the genome of anactinomycete Streptomyces lactacystinaeus, which is alactazole-producing microbe. Four copies of tRNA^(Glu) were encoded inthe genome of S. lactacystinaeus. Three of these copies were tRNA^(Glu)(CUC) having completely the same nucleotide sequence, and the other copywas tRNA^(Glu) (UUC). FIG. 5 shows the alignment of the tRNA^(Glu)nucleotide sequences of E. coli and the producing microbe. As shown inFIG. 5 , tRNA^(Glu) having the anticodon UUC had CA as the 72nd and 73rdbases, and tRNA^(Glu) having the anticodon CUC had UA as the 72nd and73rd bases.

The two types of tRNA^(Glu) were prepared, and DNA templates of the twotRNA^(Glu) (CUC and UUC) were each prepared (see FIG. 6 ). The synthesisof the DNA templates was performed by the same operation as in the DNAtemplate synthesis for the precursor peptide. Tables 17 to 22 show thereaction composition and reaction conditions of the primer extensionsystem, and the reaction composition and reaction conditions of the PCRsystem.

2 μl of the 2nd PCR products were electrophoresed to confirm that DNAtemplates having the length of interest were amplified. The 2nd PCRproducts confirmed to be amplified were subjected to phenol-chloroformextraction to remove proteins. Finally, pellets obtained by ethanolprecipitation were dissolved in 400 μl of MilliQ water.

TABLE 17 Primer extension reaction composition Taq DNA polymerase 0.75(μl) 10 × PCR Buffer 10 5 mM dNTPs 5 250 mM MgCl₂ 1.0 200 μM F1 primer0.5 200 μM R1 primer 0.5 MilliQ water 82.25 Total 100 μl

TABLE 18 Primer extension reaction conditions 94° C. 1.0 min 50° C. 1.0min {close oversize bracket} 5 cycles 68° C. 1.0 min

TABLE 19 1st PCR reaction composition Taq DNA polymerase 0.75 (μl) 10 ×PCR Buffer 10 5 mM dNTPs 5 250 mM MgCl₂ 1.0 200 μM F2 primer 0.5 200 μMR2 primer 0.5 MilliQ water 81.75 Extension product 0.5 Total 100 μl

TABLE 20 1st PCR reaction conditions 94° C. 40 min 50° C. {closeoversize bracket} 5 cycles 40 min 68° C. 40 min

TABLE 21 2nd PCR reaction composition Taq DNA polymerase 30.0 (μl) 10 ×PCR Buffer 400 5 mM dNTPs 200 250 mM MgCl₂ 40.0 50 μM F2 primer 40.0 50μM R3 primer 40.0 1st PCR product 20.0 MilliQ water 3230 Total 4000 μl

TABLE 22 2nd PCR reaction conditions 94° C. 40 min 50° C. {closeoversize bracket} 14 cycles 40 min 68° C. 40 min

(2) In Vitro Transcription Reaction

In vitro transcription reaction was performed under the reactionconditions of Table 23 below with the synthesized DNA templates. Thetranscription reaction was performed by incubation at 37° C. for 20hours.

After the completion of the in vitro transcription reaction, thetemplate DNAs were degraded by DNase treatment. Then, proteins wereremoved by isopropanol precipitation. Pellets obtained by theisopropanol precipitation were dissolved in 400 μl of MilliQ andpurified using a modified gel. The RNA solutions thus gel-purified weresubjected to ethanol precipitation again. Then, these solutions wereadjusted so as to attain 250 μM solutions while the concentration wasmeasured using Nanodrop, which were then stored at −80° C.

TABLE 23 In vitro transcription reaction composition T7 RNA polymerase80.0 (μl) 10 × T7 Buffer 400 25 mM NTPs 600 250 mM MgCl₂ 360 100 mM DTT400 2M KOH (well mixed after addition) 45.0 100 mMGMP 200 2nd PCRproduct 400 MilliQ water 1515 Total 4000 μl

(Preparation of GluRS)

Aminoacylation enzyme GluRS aminoacylates tRNA^(Glu) with glutamic acid,as shown below. Actinomycete-type GluRS was obtained as a recombinantprotein.

Since neither a strain nor genomic DNA of the lactazole-producingmicrobe S. lactacystinaeus was obtained, GluRS within the genome of S.lividans used as a host heterologously expressing lactazole was obtainedas a recombinant protein (Hayashi, S. et al. Genome mining reveals aminimum gene set for the biosynthesis of 32-membered macrocyclicthiopeptides lactazoles. Chem Biol 21, 679-688, (2014)).

Specifically, a gluRS region was first amplified by PCR using primersgluRS-Fw-NdeI and gluRS-Rv-XhoI with S. lividans genome extracts as atemplate. The amplified fragment was cloned into pET26b vector throughNdeI and XhoI to obtain gluRS/pET26b.

Next, gluRS/pET26b was transferred to E. coli BL21 (DE3) for proteinexpression by heat shock method to obtain transformants. One colonythereof was precultured at 37° C. for 16 hours using 6 ml of LB medium.4 ml of the resulting cultures was inoculated to 200 ml of LB medium.Subsequently, the bacterial cells were shake-cultured at 150 rpm at 37°C. for 2 hours and then cooled in ice. IPTG was added thereto with afinal concentration of 0.1 mM, and the bacterial cells wereshake-cultured at 180 rpm at 18° C. for 20 hours. The bacterial cellsthus cultured were centrifuged at 4720×g for 10 minutes for harvest andthen stored at −80° C. For the extraction of a recombinant protein, thebacterial cells were suspended in 50 mM Tris-HCl pH 8.0, 300 mM NaCl and20 mM imidazole pH 8.0. The suspended bacterial cells wereultrasonically disrupted, and the suspension was centrifuged at 10300×gat 4° C. for 30 minutes. From the obtained supernatant, the protein wassubjected to His-tag-purification using Ni-NTA. The column was washedwith the same buffer as used in the suspension, followed by elution witha buffer having an imidazole concentration elevated to 200 mM. Theelution solution was dialyzed overnight against Tris-HCl pH 7.5. Theactinomycete-type GluRS thus His-tag-purified was subjected to SDS-PAGE.As a result, a band corresponding to His-Tag-bound GluRS was observed ata molecular weight of approximately 55 kDa, confirming that the proteinof interest was correctly obtained. The concentration calculated fromabsorbance at UV 280 nm was 39.0 μM (approximately 2.1 mg/ml).

<Preparation of Cyclic Compound>

Cyclic compounds represented by the formula (I) were formed from theprecursor peptide obtained in the preceding section <Synthesis ofprecursor peptide>.

[Example 1] Azole Ring Formation and α,β-Unsaturated Amino AcidFormation

First, stepwise reaction was performed by allowing azole ring formationreaction to proceed completely using LazD, LazE and LazF, and after alapse of given time, adding LazB and tRNA^(Glu) to the reaction systemso that α,β-unsaturated amino acid formation reaction proceeded.

LazA* was synthesized on the scale of 5.0 μl of a cell-free translationsystem. 25 μl of a reaction solution supplemented with a coenzyme andLazD, LazE and LazF was allowed to act at 25° C. for 5 hours. After5-hour reaction, half the amount, i.e., 12.5 μl, was desalted using ziptip C18 and analyzed by MALDI-TOF MS. LazB, tRNA^(Glu) and GluRS wereadded to the remaining half (12.5 μl) and reacted again at 25° C. for 15hours.

Tables 24 and 25 show the composition of the azole ring formationreaction system and the composition of the α,β-unsaturated amino acidformation reaction system, respectively.

As a result of the analysis by MALDI-TOF MS, it was found, as shown inFIG. 7 , that the formation reaction of four azole rings almostproceeded through the 5-hour reaction mediated by LazD, LazE and LazF.When LazB, LazF, tRNA^(Glu) and GluRS were added thereto, a majorreaction product was detected at m/z 5502.5 by MALDI-TOF MS. Thisindicates that a mass shift of −90 Da occurred by the α,β-unsaturatedamino acid-forming enzymes. The dehydration of five H₂O moleculesoccurred, confirming that azole rings and dehydroalanine were formedfrom LazA*.

As described above, the in vitro reconstruction of azole ring formationand α,β-unsaturated amino acid formation in the lactazole biosynthesispathway was achieved.

TABLE 24 Azole ring formation reaction composition 5.0 μl LazA* 50 mMTris-HCl pH 8.0 5 mM ATP (adjusted to pH 7.0 with NaOH) 10 mM MgCl₂ 1 mMDTT 1 μM LazD 1 μM LazE 2 μM LazF Total volume 25 μl

TABLE 25 α,β-Unsaturated amino acid formation reaction composition 12.5μl LazA* having azole rings formed 10 μM Producing microbe tRNA^(Glu) 1μM GluRS 2 μM LazB 1 μM LazF Total volume 15 μl

[Comparative Example 1] Azole Ring Formation and α,β-Unsaturated AminoAcid Formation

The azole ring-forming enzymes LazD, LazE and LazF and theα,β-unsaturated amino acid-forming enzymes LazB and LazF were reacted atthe same time, and the reaction product was analyzed.

LazA* was synthesized on the scale of 2.5 μl of a cell-free translationsystem. LazD, LazE, LazF, LazB and the other cosubstrates were addedthereto according to the composition given below, and reacted. Thereaction was performed by incubation at 25° C. for 20 hours. After thereaction, the reaction solution was desalted using zip tip C18 andanalyzed by MALDI-TOF MS.

Table 26 shows the composition of the azole ring and α,β-unsaturatedamino acid formation reaction system.

As a result of the analysis by MALDI-TOF MS, four major reactionproducts were found at m/z 5502.6, 5656.6, 5675.6, and 5695.7, whereas apeak of the peptide of interest (m/z 5520) in which four azole rings andfour dehydroalanine moieties were formed was not confirmed. These fourobserved peaks were not consistent with any of m/z of possibleintermediates (combinations of varying numbers of azole rings formed,varying numbers of α,β-unsaturated amino acids formed and varyingnumbers of glutamylation products). Three peaks at m/z 5656.6, 5675.6and 5695.7 were presumably peaks derived from compounds resulting fromMichael addition reaction of compounds having a strongly nucleophilicthiol group or the like with dehydroalanine.

TABLE 26 Azole ring and α,β-unsaturated amino acid formation reactioncomposition 2.5 μl LazA* 50 mM Tris-HCl pH 8.0 5 mM ATP (adjusted to pH7.0 with NaOH) 10 mM MgCl₂ 1 mM DTT 1 μM LazD 1 μM LazE 2 μM LazF 10 μMProducing microbe tRNA^(Glu) 1 μM GluRS 2 μM LazB Total volume 15 μl

[Comparative Example 2] Azole Ring Formation and α,β-Unsaturated AminoAcid Formation

The stepwise reaction which involved allowing azole ring formationreaction and then α,β-unsaturated amino acid formation reaction toproceed in Example 1 was changed to a stepwise reaction which involvedallowing α,β-unsaturated amino acid formation reaction and then azolering formation reaction to proceed. Specifically, this reaction wasperformed as follows.

LazA* was synthesized on the scale of 5.0 μl of a cell-free translationsystem. LazB, tRNA^(Glu) and GluRS were added thereto and reacted at 25°C. for 5 hours. After 5-hour reaction, half the amount was desaltedusing zip tip C18 and analyzed by MALDI-TOF MS. LazD, LazE and LazF wereadded to the remaining half and reacted again at 25° C. for 15 hours.

Tables 27 and 28 show the composition of the α,β-unsaturated amino acidformation reaction system and the composition of the azole ringformation reaction system, respectively.

As a result of the analysis by MALDI-TOF MS, the dehydration reaction of1 or 2 molecules associated with α,β-unsaturated amino acid formationwas confirmed, as shown in FIG. 8 , in the 5-hour reaction mediated byLazB, LazF, tRNA^(Glu) and GluRS. When the azole ring-forming enzymesLazD, LazE and LazF were added thereto, many peaks considered as peaksof peptides differing in modified state were detected, unlike the chartshown in FIG. 7 . This means that when α,β-unsaturated amino acidformation reaction proceeds first, subsequent modification reaction doesnot proceed smoothly.

TABLE 27 α,β-Unsaturated amino acid formation reaction composition 5.0μl LazA* 50 mM Tris-HCl pH 8.0 5 mM ATP (adjusted to pH 7.0 with NaOH)10 mM MgCl₂ 1 mM DTT 10 μM Producing microbe tRNA^(Glu) 1 μM GluRS 2 μMLazB 2 μM LazF Total volume 25 μl

TABLE 28 Azole ring formation reaction composition 12.5 μl LazA* havingformed α,β-unsaturated amino acids 1 μM LazD 1 μM LazE 1 μM LazF Totalvolume 15 μl

[Example 2] Macrocyclization Reaction

As mentioned in the preceding section [Example 1], the in vitroreconstruction of azole ring formation and α,β-unsaturated amino acidformation was achieved. Subsequently, macrocyclization reaction wasperformed using a prepared recombinant enzyme. The reaction wasperformed using biosynthetic enzyme LazC presumably involved inmacrocyclization reaction.

LazA* was synthesized on the scale of 5.0 μl of a cell-free translationsystem. LazD, LazE, LazF, LazB, LazC and other cosubstrates were addedthereto according to the composition given below, and reacted in 30 μlin total of a reaction system. The reaction was performed in reactionsystems including four reaction systems: A. a reaction system of LazA*alone; B. LazA*+LazD, LazE and LazF; C. LazA*+LazD, LazE, LazF, LazB,tRNA^(Glu) and GluRS; and D. LazA*+LazD, LazE, LazF, LazB, tRNA^(Glu),GluRS and LazC. Each reaction was performed by incubation at 25° C. for20 hours. The composition of the reaction systems is shown in Table 29.

After the reaction, each reaction solution was desalted using zip tipC18 and analyzed by MALDI-TOF MS. For the analysis, half the amount,i.e., 15 μl, was analyzed using sinapic acid (SA) as a matrix, and theremaining half was analyzed using CHCA (α-cyano-4-hydroxycinnamic acid)as a matrix. The samples with sinapic acid used as a matrix weremeasured in a region of molecular weights of 2000 or larger. The sampleswith CHCA used as a matrix were measured in a region of molecularweights of 3000 or smaller.

The results of the analysis by MALDI-TOF MS are shown in FIG. 9 . Asshown in FIG. 9 , peptides in a region from m/z 5400 to 5800 disappearedin the system supplemented with LazC, and a peptide of m/z 4102.1accumulated instead, demonstrating that LazC consumed the substrate. Thesignal of m/z 4102.1 was consistent with m/z of a leader peptide cleavedoff by LazC (calcd. m/z 4102.1). This peak of m/z 4102.1 was furtheranalyzed by MALDI-TOF MS/MS. As a result, this peak was found to bederived from the leader peptide.

As shown in FIG. 10 , a peak consistent with m/z of a Na adduct oflactazole A (calcd. 1423.369) was confirmed in the system of LazC(+) bythe comparison of mass spectra between LazC(+) and LazC(−). It was alsoconfirmed by LC-MS and LC/MS/MS that a cyclized compound was able to besynthesized as the enzymatic reaction product.

Since m/z of a leader peptide is easily detected, this m/z can serve asan index for conveniently determining whether a macrocyclization producthas been formed.

As described above, a method for synthesizing a cyclized compound usingenzymes for lactazole A in vitro was established.

TABLE 29 Azole ring, α,β-unsaturated amino acid, and macrocycleformation reaction composition 5.0 μl LazA* 50 mM Tris-HCl pH 8.0 5 mMATP (adjusted to pH 7.0 with NaOH) 10 mM MgCl₂ 1 mM DTT 1 μM LazD 1 μMLazE 2 μM LazF 10 μM Producing microbe tRNA^(Glu) (CUC) 1 μM GluRS 2 μMLazB 2 μM LazC Total volume 30 μl

<Substrate Tolerance>

The constructed method for synthesizing a cyclized compound usingenzymes for lactazole A in vitro as mentioned above was examined forapplicable substrate tolerance.

[Example 3] Substrate Tolerance to Length of Precursor Peptide-1

In order to confirm the possibility of forming macrocycles other than a32-membered ring by the macrocyclase LazC, precursor peptides insertedwith Ala were prepared by inserting Ala to the core peptide region.Macrocyclization was performed with these precursor peptides assubstrates.

As in Example 1, a mutant DNA template was prepared by primer extensionand PCR and translated in a cell-free translation system to confirm thatthe precursor peptides of interest, inserted with Ala, were synthesized.Subsequently, LazD, LazE, LazF, LazB, tRNA^(Glu) (CUC), GluRS and LazCwere added to respective translationally-synthesized Ala-insertedprecursor peptides and reacted. Table 30 shows the composition of azolering, α,β-unsaturated amino acid and macrocycle formation reaction. Thereaction was performed by incubation at 25° C. for 20 hours. Thereaction product was analyzed by MALDI-TOF MS and LC-MS.

Table 31 shows the sequences of the prepared precursor peptides and thenumbers of azole rings and α,β-unsaturated amino acids formed observedin each precursor peptide. Each of the precursor peptides exhibited thenumbers of azole rings and α,β-unsaturated amino acids formed similar tothose of the native one (LazA*). This demonstrated that the insertion ofAla hardly influences azole ring formation and α,β-unsaturated aminoacid formation. In the table, symbols such as “4, 5” represent that twopeaks have almost the same intensity. The same holds true for suchsymbols in the tables given below.

FIG. 11 shows mass chromatograms about the leader peptide cleaved offthrough LazC reaction which is a reaction product of macrocycleformation reaction. The leader peptides are basically cleaved offspecifically for the macrocyclization reaction of LazC. Provided thationic strengths in the chromatograms were not saturated and fell withina dynamic range, it was believed that the peak intensity of each leaderpeptide cleaved off was proportional to macrocyclization efficiency.Accordingly, the peak areas of the eliminated leader peptides forrespective analogs are summarized in FIG. 12 . As is evident from FIGS.11 and 12 , LazC was found to tolerate ring expansion.

TABLE 30 Azole ring, α,β-unsaturated amino acid, and macrocycleformation reaction composition 5.0 μl LazA* 50 mM Tris-HCl pH 8.0 5 mMATP (adjusted to pH 7.0 with NaOH) 10 mM MgCl₂ 1 mM DTT 1 μM LazD 1 μMLazE 2 μM LazF 10 μM Producing microbe tRNA^(Glu)(CUC) 1 μM GluRS 2 μMLazB 2 μM LazC Total volume 30 μl

TABLE 31 The number of The number of Amino acid sequence azole moietiesdehydroalanine Analog name 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17formed moieties formed Native S W G S C S C Q A S S S C A Q P 4 5 1A2 SA W G S C S C Q A S S S C A Q P 4 5 2A3 S W A G S C S C Q A S S S C A QP 4 5 3A4 S W G A S C S C Q A S S S C A Q P 4 5 4A5 S W G S A C S C Q AS S S C A Q P 4, 5 5 5A6 S W G S C A S C Q A S S S C A Q P 4 5 6A7 S W GS C S A C Q A S S S C A Q P 4 5 7A8 S W G S C S C A Q A S S S C A Q P 45 8A9 (9A10) S W G S C S C Q A A S S S C A Q P 3, 4 5 10A11 S W G S C SC Q A S A S S C A Q P 3, 4 5 11A12 S W G S C S C Q A S S A S C A Q P 4 512A13 S W G S C S C Q A S S S A C A Q P 4 5

[Example 3] Substrate Tolerance to Length of Precursor Peptide-2

Most of the Ala-inserted precursor peptides in which one residue of Alawas inserted became a macrocyclic compound having an expanded ring inresponse to macrocyclization reaction mediated by LazC. Accordingly, inorder to examine how large the ring formed by LazC could be or how smallthe ring formed by LazC could be, mutant precursor peptides wereprepared by inserting two or three residues of Ala or by deleting one ortwo residues of amino acids from LazA*. These mutant precursor peptideswere reacted as substrates with LazC.

In accordance with the method of Example 2, the precursor peptides shownin Table 32 were prepared, and each precursor peptide was subjected toazole ring, α,β-unsaturated amino acid and macrocycle formationreaction. FIG. 13 shows the chromatograms of leader peptides cleaved offby the macrocyclization of the analogs. FIG. 14 shows the peak areas ofthe leader peptides.

TABLE 32 Amino acid sequence Analog name 1 2 3 4 5 6 7 8 9 10 11 12 1314 15 16 17 18 19 LazA* S W G S C S C Q A S S S C A Q P 2AA3 S W A A G SC S C Q A S S S C A Q P 8AA9 S W G S C S C Q A A A S S S C A Q P 2A3 +8A9 S W A G S C S C Q A A S S S C A Q P 2AAA3 S W A A A G S C S C Q A SS S C A Q P 8AAA9 S W G S C S C Q A A A A S S S C A Q P 3G_deletion S WS C S C Q A S S S C A Q P 9A_deletion S W G S C S C Q S S S C A Q P3G9A_deletion S W S C S C Q S S S C A Q P

As seen from Examples 3 and 4, LazC successfully formed macrocycles from26-membered to 41-membered rings, demonstrating that LazC has substratetolerance to the size of a macrocycle.

[Example 5] Ser(4), Cys(5), Ser(6), and Cys(7)-Substituted Analog

The 4SCSC7_4AAAA7 precursor peptide was prepared by substituting all theamino acid residues Ser(4), Cys(5), Ser(6) and Cys(7), which areconverted to azole rings and α,β-unsaturated amino acids, by Ala. Thisprecursor peptide was reacted as a substrate with LazC.

In according with the method of Example 3, a mutant DNA templateencoding 4SCSC7_4AAAA7 was prepared and translated in the cell-freetranslation system to confirm that the mutant precursor peptide ofinterest was synthesized. Subsequently, LazD, LazE, LazF, LazB,tRNA^(Glu) (CUC), GluRS and LazC were added to thetranslationally-synthesized mutant precursor peptide and reacted. Thereaction product was analyzed by MALDI-TOF MS and LC-MS.

The amino acid sequence of the prepared 4SCSC7_4AAAA7 precursor peptideis shown in Table 33.

FIG. 15 shows the chromatogram of a leader peptide in the reactionproduct. Leader peptide elimination was also seen in the 4SCSC7_4AAAA7precursor peptide at a level equivalent to LazA*, demonstrating thatSer(4), Cys(5), Ser(6) and Cys(7) are not important for the substraterecognition of LazC.

TABLE 33 Amino acid sequence Analog name 1 2 3 4 5 6 7 8 9 10 11 12 1314 15 16 LazA* S W G S C S C Q A S S S C A Q P 4SCSC7_4AAAA7 S W G A A AA Q A S S S C A Q P

[Example 6] Ser(11), Ser(12), Cys(13) or Ser(10)-Substituted Analog

Mutant precursor peptides were prepared by substituting each of Ser(11),Ser(12), Cys(13) and their neighboring Ser(10) one by one by Ala. Thesemutant precursor peptides were analyzed for whether to be recognized asa substrate by LazC.

In according with the method of Example 3, mutant DNA templates encodingrespective analogs described in Table 34 were prepared and translated ina cell-free translation system to confirm that the mutant precursorpeptides of interest were synthesized. Subsequently, LazD, LazE, LazF,LazB, tRNA^(Glu) (CUC), GluRS and LazC were added to eachtranslationally-synthesized mutant precursor peptide and reacted. Thereaction products were analyzed by MALDI-TOF MS and LC-MS.

Table 34 shows the amino acid sequences of the prepared precursorpeptides, and the numbers of azole rings and α,β-unsaturated amino acidsformed.

FIG. 16 shows the chromatograms of leader peptides in the reactionproducts. A leader peptide cleaved off was slightly seen in S11A andC13A, whereas such a leader peptide was hardly seen in S11A and S12A.This demonstrated that for the recognition of a substrate by LazC,Ser(11) and Ser(12) are important and Ser(10) and Cys(13) are alsorelatively important.

TABLE 34 The number The of number dehydro- of azole alanine Amino acidsequence moieties moieties Analog name 1 2 3 4 5 6 7 8 9 10 11 12 13 1415 16 formed formed LazA*

W G S

Q A

A Q P 4 5 4SCSC7_4AAAA7

W G A A A A Q A

A Q P 2 3 4SCSC7_4AAAA7S10A S W G A A A A Q A A S S C A Q P 2 24SCSC7_4AAAA7S11A S W G A A A A Q A S A S C A Q P 1 3 4SCSC7_4AAAA7S12AS W G A A A A Q A S S A C A Q P 2 2 4SCSC7_4AAAA7C13A S W G A A A A Q AS S S A A Q P 1 3

[Example 7] Trp(2), Gly(3), Gln(8), Ala(9), Gln(15) orPro(16)-Substituted Analog

Example 5 demonstrated that macrocyclization proceeds even if Ser(4),Cys(5), Ser(6) and Cys(7) are substituted. Example 6 demonstrated thatfor the recognition of a substrate by LazC, Ser(11) and Ser(12) areimportant and Ser(10) and Cys(13) are also relatively important.

The other amino acids Trp(2), Gly(3), Gln(8) and Ala(9) supposed to becontained in the macrocycle were studied for whether to besubstitutable. Also, amino acids exterior to the macrocycle were studiedfor whether macrocyclization would also proceed in Gln(15) orPro(16)-substituted analogs.

In according with the method of Example 3, mutant DNA templates encodingrespective precursor peptides described in Table 35 were prepared andtranslated in the cell-free translation system to confirm that themutant precursor peptides of interest were synthesized. Subsequently,LazD, LazE, LazF, LazB, tRNA^(Glu) (CUC), GluRS and LazC were added toeach translationally-synthesized mutant precursor peptide and reacted.The reaction product were analyzed by MALDI-TOF MS and LC-MS. Theresults of the LC-MS analysis (MS peaks of leader peptides andmacrocyclization products) are shown in FIGS. 17, 18 and 19 .

In the table, the symbol “( )” in “3, (2)”, etc. represents a minorpeak. The same holds true for such symbols in the tables given below.

Results about the reaction efficiency in substitution of theintra-macrocycle residues (Trp(2), Gly(3), Gln(8), Ala(9), Gln(15) andPro(16)) are summarized in FIG. 20 . In FIG. 20 , the amino acidresidues with parentheses mean residues having reduced reactionefficiency.

TABLE 35 Analog name 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Azole ringDehydroalanine 4SCSC7_4AAAA7_W2A S A G A A A A Q A S S S C A Q P 2 3(2)4SCSC7_4AAAA7_W2E S E G A A A A Q A S S S C A Q P 2 3 4SCSC7_4AAAA7_W2RS R G A A A A Q A S S S C A Q P 2 3 4SCSC7_4AAAA7_W2S S S G A A A A Q AS S S C A Q P 2 4, 3 4SCSC7_4AAAA7_W2V S V G A A A A Q A S S S C A Q P 23(2) 4SCSC7_4AAAA7_W2F S F G A A A A Q A S S S C A Q P 2 34SCSC7_4AAAA7_G3E S W E A A A A Q A S S S C A Q P 2 3 4SCSC7_4AAAA7_G3RS W R A A A A Q A S S S C A Q P 2 3 4SCSC7_4AAAA7_G3V S W V A A A A Q AS S S C A Q P 2 3 4SCSC7_4AAAA7_G3S S W S A A A A Q A S S S C A Q P 2 4,3 4SCSC7_4AAAA7_G3F S W F A A A A Q A S S S C A Q P 2 34SCSC7_4AAAA7_G3W S W W A A A A Q A S S S C A Q P 2 3 4SCSC7_4AAAA7_Q8AS W G A A A A A A S S S C A Q P 1(2) 3 4SCSC7_4AAAA7_Q8E S W G A A A A EA S S S C A Q P 1 4 4SCSC7_4AAAA7_Q8R S W G A A A A R A S S S C A Q P 1,2 3 4SCSC7_4AAAA7_Q8V S W G A A A A V A S S S C A Q P 1, 2 34SCSC7_4AAAA7_Q8S S W G A A A A S A S S S C A Q P 1, 2(3) 3(azole 3 +Dha3) 4SCSC7_4AAAA7_Q8F S W G A A A A F A S S S C A Q P 2 34SCSC7_4AAAA7_Q8W S W G A A A A W A S S S C A Q P 2 3 4SCSC7_4AAAA7_A9ES W G A A A A Q E S S S C A Q P 1(2) 0 4SCSC7_4AAAA7_A9R S W G A A A A QR S S S C A Q P 2, 1 3 4SCSC7_4AAAA7_A9V S W G A A A A Q V S S S C A Q P2, 1 3 4SCSC7_4AAAA7_A9S S W G A A A A Q S S S S C A Q P 2 44SCSC7_4AAAA7_A9F S W G A A A A Q F S S S C A Q P 2, 1 34SCSC7_4AAAA7_A9W S W G A A A A Q W S S S C A Q P 2 3 4SCSC7_4AAAA7_Q15AS W G A A A A Q A S S S C A A P 2 3 4SCSC7_4AAAA7_P16A S W G A A A A Q AS S S C A Q A 2 3 4SCSC7_4AAAA7_15QP16_15AA16 S W G A A A A Q A S S S CA A A 2 3

[Example 8-1] Substrate Tolerance to Length of Precursor Peptide-3

In order to further study the size of the macrocycle that could becatalyzed by LazC, Ala multiply-inserted precursor peptides wereprepared by inserting multiple Ala residues to the 4SCSC7_4AAAA7precursor peptide, whereas multiply-deleted precursor peptides wereprepared by deleting multiple residues therefrom.

In according with the method of Example 3, mutant DNA templates encodingrespective precursor peptides with one to seven Ala residues inserted tothe 4SCSC7_4AAAA7 precursor peptide and precursor peptides with one toeight amino acid residues deleted from the 4SCSC7_4AAAA7 precursorpeptide were prepared and translated in the cell-free translation systemto confirm that the mutant precursor peptides of interest weresynthesized. Subsequently, LazD, LazE, LazF, LazB, tRNA^(Glu) (CUC),GluRS and LazC were added to each translationally-synthesized mutantprecursor peptide and reacted. The reaction product was analyzed byMALDI-TOF MS and LC-MS.

Table 36 shows the amino acid sequences of the prepared Alamultiply-inserted and multiply-deleted precursor peptides, and thenumbers of azole rings and α,β-unsaturated amino acids formed. Similarazole ring formation and α,β-unsaturated amino acid formation were seenin the mutant precursor peptides, demonstrating that LazD, LazE, LazFand LazB tolerate these mutant precursor peptides as substrates. Thissuggests that for the substrate recognition of the azole ring-formingenzymes and the α,β-unsaturated amino acid-forming enzymes, distancefrom a leader peptide region is not important and the arrangement of anamino acid sequence surrounding the residues serving as a substrate isimportant.

FIGS. 21 and 22 show the chromatograms of the leader peptides in thereaction products. FIGS. 23 and 24 show the peak areas of the leaderpeptides.

TABLE 36 The The number number of dehydro- of azole alanine Amino acidsequence rings moieties Analog name 1 2 3 4 5 6 7 8 9 10 11 12 13 14 1516 17 18 19 20 21 22 23 formed formed LazA*

W G S

Q A

A Q P 4 4 4SCSC7_4AAAA7

W G A A A A Q A

A Q P 2 3 4SCSC7_4AAAA7_1×Ala insertion

W G A A A A A Q A S S

C A Q P 2, (1) 3 4SCSC7_4AAAA7_2×Ala insertion

W G A A A A A A Q A S S

C A Q P 2, (1) 3 4SCSC7_4AAAA7_3×Ala insertion

W G A A A A A A A Q A S S

C A Q P 2, (1) 3 4SCSC7_4AAAA7_4×Ala insertion

W G A A A A A A A A Q A S S

C A Q P 2, (1) 3 4SCSC7_4AAAA7_5×Ala insertion

W G A A A A A A A A A Q A S S

C A Q P 2, (1) 3 4SCSC7_4AAAA7_6×Ala insertion

W G A A A A A A A A A A Q A S S

C A Q P 2, 1 3 4SCSC7_4AAAA7_7×Ala insertion

W G A A A A A A A A A A A Q A S S

C A Q P 2, (1) 3 4SCSC7_4AAAA7_1×Ala deletion

W G A A A Q A S S

C A Q P 2 3, (2) 4SCSC7_4AAAA7_2×Ala deletion

W G A A Q A S S

C A Q P 2 3, (2) 4SCSC7_4AAAA7_3×Ala deletion

W G A Q A S S

C A Q P 2 3 4SCSC7_4AAAA7_4×Ala deletion

W G Q A S S

C A Q P 2 3, (2) 4SCSC7_4AAAA7_4×Ala_1 × Gly

W Q A S S

C A Q P 2 2, (3) deletion 4SCSC7_4AAAA7_4×Ala_1 × Gly, Gln

W A S S

C A Q P 2 2, 3 deletion 4SCSC7_4AAAA7_5×Ala_1 × Gly, Gln

W S S

C A Q P 2, (1) 3 deletion 4SCSC7_4AAAA7_5×Ala_1 × Gly, Gln,

S S

C A Q P 2, 1 3, (2) Trp deletion

[Example 8-2] Substrate Tolerance to Length of Precursor Peptide-4

In order to further study the size of the macrocycle that could beformed by the Laz enzyme group, Ala and Asn multiply-inserted precursorpeptides were prepared by inserting multiple Ala and Asn residues to the4SCSC7_4AAAA7 precursor peptide, whereas multiply-deleted precursorpeptides were prepared by deleting multiple residues therefrom.

In according with the method of Example 3, mutant DNA templates encodingrespective precursor peptides with Ala and Asn inserted to the4SCSC7_4AAAA7 precursor peptide and precursor peptides with amino acidresidues deleted from the 4SCSC7_4AAAA7 precursor peptide were preparedand translated in the cell-free translation system to confirm that themutant precursor peptides of interest were synthesized. Subsequently,LazD, LazE, LazF, LazB, tRNA^(Glu) (CUC), GluRS and LazC were added toeach translationally-synthesized mutant precursor peptide and reacted.The reaction products were analyzed by LC-MS. In this experiment,modification reaction mediated by the Laz enzyme group and the formationof a macrocyclization product were confirmed by broad extracted ionchromatograms (brEIC) in which peak(s) detected in the range of ±400 or±600 of the tetravalent or pentavalent m/z value of a precursor peptidewere integrated. In the brEIC, the presence of a precursor peptide, alinear intermediate peptide having azoline/azole/dehydrated amino acidand a leader peptide fragment (LP-NH₂) through LazC reaction can beconfirmed at the same time. FIG. 25 shows brEIC of each reactionproduct.

[Example 9-1] Substrate Tolerance of Azole Ring-Forming Enzyme toCys-Substituted Analog

In order to determine the substrate recognition of the azolering-forming enzymes, Cys-substituted precursor peptides were preparedby substituting residues of the core peptide region one by one by Cys,and reacted with the azole ring-forming enzymes.

In according with the method of Example 3, mutant DNA templates encodingrespective Cys-substituted precursor peptides with amino acid residuesof the core peptide region substituted one by one by Cys were preparedand translated in the cell-free translation system to confirm that themutant precursor peptides of interest were synthesized. Subsequently,LazD, LazE and LazF were added to each translationally synthesizedmutant precursor peptide and reacted. The reaction products wereanalyzed by MALDI-TOF MS.

Table 37 shows the amino acid sequences of the prepared Cys-substitutedprecursor peptides, and the observed number of azole rings formed. Theazole ring-forming enzymes were found to recognize a region rich in Cysor Ser and tend to cause the azole cyclization of Cys or Ser residues inthis region.

TABLE 37 Amino acid sequence The number of azole Analog name 1 2 3 4 5 67 8 9 10 11 12 13 14 15 16 moieties formed Native

W G S

Q A

A Q P 4 S1C C W G S C S C Q A S S S C A Q P 4 W2C S C G S C S C Q A S SS C A Q P 4 G3C S W C S C S C Q A S S S C A Q P 4 S4C S W G C C S C Q AS S S C A Q P 4 S6C S W G S C C C Q A S S S C A Q P 4 Q8C S W G S C S CC A S S S C A Q P 4, 5 A9C S W G S C S C Q C S S S C A Q P 4, 5 S10C S WG S C S C Q A C S S C A Q P 5, (4) S11C S W G S C S C Q A S C S C A Q P4 S12C S W G S C S C Q A S S C C A Q P 4 A14C S W G S C S C Q A S S S CC Q P 4 Q15C S W G S C S C Q A S S S C A C P 4 P16C S W G S C S C Q A SS S C A Q C 4

[Example 9-2] Substrate Tolerance of Macrocyclization to Ala-SubstitutedAnalog

As in Example 1, mutant DNA templates were prepared by primer extensionand PCR and translated in the cell-free translation system to synthesizeS1A, W2A, G3A, S4A, C5A, S6A, C7A, Q8A, S10A, S11A, S12A, C13A, Q15A andP16A altered precursor peptides (see FIG. 26 ) in which the 1st to 16thnative amino acids, respectively, were replaced with Ala.

Subsequently, each altered precursor peptide mentioned above was reactedas in Example 8-2, and the reaction product was analyzed by LC-MS.Modification reaction mediated by the Laz enzyme group and the formationof macrocyclization products were confirmed by broad extracted ionchromatograms (brEIC) in which peaks detected in the range of ±400 ofthe tetravalent m/z value of a precursor peptide were integrated. FIG.26 shows brEIC of each reaction product. In the chromatograms, no peakof a macrocyclization product was observed for S1A, S11A, and S12A.

[Example 9-3] Substrate Tolerance of Macrocyclization to Lys-SubstitutedAnalog

As in Example 1, mutant DNA templates were prepared by primer extensionand PCR and translated in the cell-free translation system to synthesizeW2K, G3K, A4K, A5K, A6K, A7K, Q8K, A9K, Q15K and P16K altered precursorpeptides (see FIG. 27 ) in which the 2nd to 9th and 14th to 16th nativeamino acids, respectively, were replaced with Lys.

Subsequently, each altered precursor peptide mentioned above was reactedin accordance with the procedures described in Example 9-2, and thereaction product was analyzed by LC-MS. Modification reaction mediatedby the Laz enzyme group and the formation of macrocyclization productswere confirmed by broad extracted ion chromatograms (brEIC) in whichpeaks detected in the range of ±400 of the tetravalent m/z value of aprecursor peptide were integrated. FIG. 27 shows brEIC of each reactionproduct.

[Example 9-4] Substrate Tolerance of Macrocyclization to Glu-SubstitutedAnalog

As in Example 9-3, W2E, G3E, A4E, A5E, A6E, A7E, Q8E, A9E, Q15E and P16Ealtered precursor peptides (see FIG. 28 ) were synthesized by replacingthe 2nd to 9th, 15th, and 16th native amino acids, respectively, withGlu. Subsequently, each altered precursor peptide mentioned above wasreacted in accordance with the procedures described in Example 9-2, andthe reaction product was analyzed by LC-MS. Modification reactionmediated by the Laz enzyme group and the formation of macrocyclizationproducts were confirmed by broad extracted ion chromatograms (brEIC) inwhich peaks detected in the range of ±400 of the tetravalent m/z valueof a precursor peptide were integrated. FIG. 28 shows brEIC of eachreaction product.

[Example 10-1] Substrate Tolerance of Macrocyclization to Ser(10),Ser(11), or Cys(13)-Substituted Analog

As in Example 1, mutant DNA templates were prepared by primer extensionand PCR and translated in the cell-free translation system to confirmthat the S10T, S11T, S11C, C13S and C13T altered precursor peptides ofinterest (see Table 38) were synthesized.

Subsequently, LazD, LazE, LazF, LazB, tRNA^(Glu) (CUC), GluRS and LazCwere added to each of the translationally-synthesized S10T, S11T, S11C,C13S and C13T altered precursor peptides and reacted. Table 39 shows thecomposition of azole ring, α,β-unsaturated amino acid and macrocycleformation reaction. The reaction was performed by incubation at 25° C.for 20 hours. The reaction products were analyzed by MALDI-TOF MS andLC-MS. FIG. 29 shows the chromatograms of leader peptides in thereaction product.

TABLE 38 Name 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 4SCSC7_4AAAA7 S W GA A A A Q A S S S C A Q P 4SCSC7_4AAAA7_S10T S W G A A A A Q A T S S C AQ P 4SCSC7_4AAAA7_S11T S W G A A A A Q A S T S C A Q P4SCSC7_4AAAA7_S11C S W G A A A A Q A S C S C A Q P 4SCSC7_4AAAA7_C13S SW G A A A A Q A S S S S A Q P 4SCSC7_4AAAA7_C13T S W G A A A A Q A S S ST A Q P

TABLE 39 Azole ring, α,β-unsaturated amino acid, and macrocycleformation reaction composition 5.0 μl LazA* 50 mM Tris-HCl pH 8.0 5 mMATP (adjusted to pH 7.0 with NaOH) 10 mM MgCl₂ 1 mM DTT 1 μM LazD 1 μMLazE 2 μM LazF 10 μM Producing microbe tRNA^(Glu) (CUC) 1 μM GluRS 2 μMLazB 2 μM LazC Total volume 30 μl

[Example 10-2] Substrate Tolerance of Macrocyclization to Analog(Substitution and Extension) as to C-Terminal Region

As in Example 1, mutant DNA templates were prepared by primer extensionand PCR and translated in the cell-free translation system to obtain theprecursor peptides altered in a C-terminal region shown in FIG. 30 .Subsequently, each of the translationally-synthesized precursor peptidesaltered in a C-terminal region was reacted in accordance with theprocedures described in Example 10-1, and the reaction product wasanalyzed by LC-MS. Modification reaction mediated by the Laz enzymegroup and the formation of macrocyclization products were confirmed bybroad extracted ion chromatograms (brEIC) in which peaks detected in therange of ±400 of the pentavalent m/z value of a precursor peptide wereintegrated. FIG. 30 shows brEIC of each reaction product.

[Example 10-3] Substrate Tolerance of Macrocyclization to Analog HavingRandom Artificial Sequence as Intra-Ring Sequence, and Analog HavingRandomized Intra-Ring Sequence and Having Extended C-Terminal Region

As in Example 1, mutant DNA templates were prepared by primer extensionand PCR and translated in the cell-free translation system to obtain thealtered precursor peptides of interest having a randomized intra-ringsequence (FIG. 31 a ), and the altered precursor peptides of interesthaving a randomized intra-ring sequence and having an extendedC-terminal region (FIG. 31 b ), as shown in FIG. 31 . Subsequently,LazD, LazE and LazF were added to each of thetranslationally-synthesized precursor peptide altered in a C-terminalregion, and reacted at 25° C. for 6 hours. Then, LazB, cosubstratetRNA^(Glu), aminoacylation enzyme GluRS and LazC were added thereto andreacted at 25° C. for 12 hours. The reaction products were analyzed byLC-MS. Modification reaction mediated by the Laz enzyme group and theformation of macrocyclization products were confirmed by broad extractedion chromatograms (brEIC) in which peaks detected in the range of ±600of the tetravalent or pentavalent m/z value of a precursor peptide wereintegrated. FIG. 31 shows brEIC of each reaction product.

[Example 11] Recognition of Leader Peptide

In order to determine the role of leader peptides in the lactazolebiosynthetic enzymes, leader peptide-truncated precursor peptides wereprepared and reacted as substrates with each modifying enzyme.

Mutant precursor peptides were prepared by N-terminally truncating aleader peptide having 38 residues. The prepared mutant precursorpeptides contained leader peptides having 30, 25, 20 and 15 residues andwere designated as LP-30, LP-25, LP-20, and LP-15, respectively. Azolering formation reaction, α,β-unsaturated amino acid formation reactionand macrocyclization reaction were performed with these four mutantprecursor peptides as substrates. The reaction products were analyzed byMALDI-TOF MS and LC-MS to confirm whether the reaction has proceeded ornot.

The amino acid sequences of the prepared leader peptide-truncatedprecursor peptides are shown in Table 40. The sequence SWGSCSCQASSSCAQPcorresponds to the core peptide region. The N terminus is alwaysformylmethionine because of synthesis by translation. As a result of theazole ring formation reaction mediated by LazD, LazE and LazF and theα,β-unsaturated amino acid formation reaction mediated by LazB and LazFwith each of the mutant precursor peptides as a substrate, it was foundthat the azole ring formation and the α,β-unsaturated amino acidformation proceeded almost completely up to LP-25, whereas theefficiency of azole ring formation and α,β-unsaturated amino acidformation was reduced in LP-20. The results about mass chromatograms areshown in FIGS. 32 to 36 .

FIG. 37 shows the mass chromatograms of leader peptides resulting frommacrocyclization reaction mediated by LazC with each of the mutantprecursor peptides as a substrate.

These results demonstrated that an important site of a leader peptidefor the azole ring-forming enzymes and the α,β-unsaturated aminoacid-forming enzymes in lactazole biosynthesis is a C-terminal side fromLeu(−25). The results also demonstrated that as for the macrocyclase,reaction proceeds in the presence of at least a C-terminal region fromLeu(−20).

TABLE 40 Analog name Amino acid sequence LazA*MSDITASRVESLDLQDLDLSELTVTSLRDTVALPENGASWGSCSCQASSSCAQP LP-30       MVESLDLQDLDLSELTVTSLRDTVALPENGASWGSCSCQASSSCAQP LP-25            MLQDLDLSELTVTSLRDTVALPENGASWGSCSCQASSSCAQP LP-20                 MLSELTVTSLRDTVALPENGASWGSCSCQASSSCAQP LP-15                      MVTSLRDTVALPENGASWGSCSCQASSSCAQP

[Example 12] Ala Scanning Experiment of Leader Peptide

Altered precursor peptides were prepared by substituting each of theamino acid residues positioned from −1 to −25 of the leader peptide inLP-25 shown in FIG. 38 by Ala to confirm substrate tolerance thereof toazole ring, α,β-unsaturated amino acid and macrocycle formationreaction.

Specifically, as in Example 1, mutant DNA templates were prepared byprimer extension and PCR and translated in the cell-free translationsystem to confirm that the altered precursor peptides of interest weresynthesized as altered forms of the leader peptide site.

Subsequently, LazD, LazE, LazF, LazB, tRNA^(Glu) (CUC), GluRS and LazCwere added to each translationally-synthesized Ala-inserted precursorpeptide and reacted. Table 41 shows the composition of azole ring,α,β-unsaturated amino acid and macrocycle formation reaction. Thereaction was performed by incubation at 25° C. for 20 hours. Thereaction products were analyzed by MALDI-TOF MS and LC-MS.

FIG. 39 shows the chromatograms of leader peptides in the reactionproducts of the altered precursor peptides prepared by the substitutionof each of the amino acid residues positioned from −1 to −25. In FIG. 39, for example, “LP-25_S-19A” means an “altered precursor peptide inwhich the amino acid position at −19 (S) was substituted by Ala”. Thesame holds true for the other altered precursor peptides.

TABLE 41 Azole ring, α,β-unsaturated amino acid, and macrocycleformation reaction composition 5.0 μl LazA* 50 mM Tris-HCl pH 8.0 5 mMATP (adjusted to pH 7.0 with NaOH) 10 mM MgCl₂ 1 mM DTT 1 μM LazD 1 μMLazE 2 μM LazF 10 μM Producing microbe tRNA^(Glu) (CUC) 1 μM GluRS 2 μMLazB 2 μM LazC Total volume 30 μl

[Example 13] Preparation and Screening of Compound Library

As shown in FIG. 40 , compound libraries were designed which wereconstituted by peptides containing “leader peptide -Ser- any amino acidsequence consisting of 5 to 10 residues -Ser-Ser-Ser-Cys- any amino acidsequence consisting of 3 residues”. DNA libraries were prepared on thebasis of the design of the compound libraries described above. Thepreparation of the DNA libraries conformed to the method described inChemistry & Biology 18, 1562-1570 (2011), and Chemistry & Biology 21,766-774 (2014). The DNA libraries were transcribed in vitro to preparemRNA libraries. It was confirmed that the mRNA libraries were able to besynthesized as shown in the electrophoretic gel of FIG. 41 . In thedrawing of the electrophoretic gel, 6 to 11 denote mRNA librariescontaining any amino acid sequence consisting of 5, 6, 7, 8, 9 and 10residues, respectively. In order to confirm that the prepared mRNAlibraries had a correct sequence, the mRNA libraries werereverse-transcribed. Then, the obtained DNA sequences were analyzedusing a high-throughput sequencer (MiSeq, Illumina K.K.). From resultsof sequencing quality score Q (FIG. 42 ) in the DNA sequencing,frameshift analysis (FIG. 43 ), sequence conservation plot (FIG. 44 )and amino acid frequency analysis (FIG. 45 ), it was confirmed that themRNA libraries were correctly constructed as designed.

According to the scheme shown in FIG. 46 , peptide-mRNA complexlibraries were constructed from the mRNA libraries mentioned above, andcompound libraries containing thiopeptides were constructed by theaction of LazB-F enzyme. Then, the compound libraries were subjected toa step of screening for a cyclic peptide interacting with enzyme iPGM.As seen from the graph of the rate of cDNA recovery shown in FIG. 47 ,cyclic peptides binding to iPGM were able to be obtained.

These results showed that the production method of the present inventionincluding using an azole ring-forming enzyme, an α,β-unsaturated aminoacid-forming enzyme and a macrocyclase can provide a compound library.

[Example 14-1] Synthesis of Cyclic Peptide Containing Non-ProteinogenicAmino Acid

On the basis of the reprogramming scheme shown in FIG. 48 ,non-proteinogenic amino acids were introduced to the core peptidemoieties of precursor peptides before azole ring formation,α,β-unsaturated amino acid formation and macrocyclization. FIG. 49 showsDNA templates and peptides formed from the templates. In FIG. 49 , theboxed codons and amino acids refer to codons and amino acids to bereprogrammed, and are positions for the introduction ofnon-proteinogenic amino acids.

In accordance with the method described in Example 1, each DNA templatedescribed above was translated and further induced into a cyclic peptidethrough the step of allowing LazB, LazC, LazD, LazE and LazF enzymes toact. The products after translation and cyclic peptide-formation weremeasured by LC/MS to confirm whether the translation for the DNAtemplates harboring the non-proteinogenic amino acids have proceeded,and whether cyclic peptides have been formed. The results are shown inTables 42 to 44. In the tables, m/z shown in the column “Observationresults” is the observed value of MS, or the value of MS of the cyclicpeptide.

The structures of the introduced non-natural amino acids are asdescribed below.

In Table 43, v.064 and v.082 denote precursor peptides obtained from DNAtemplates containing no codon to be reprogrammed. MS of a cyclic peptidewas also observed in v.064 and v.082. The obtainment of the cyclicpeptides from v.064 and v.082 means that the peptides corresponding to Zand Z′ in the formula (II) and the formula (III) are not limited bytheir lengths or types.

TABLE 42 DNA Codon template reprogrammingAmino acid sequence of precursor peptide v.061 tgg = WMSDITASRVESLDLQDLDLSELTVTSLRDTVALPENGASWGAAAAQASSSCAQP v.061tgg = N^(Me)Gly MSDITASRVESLDLQDLDLSELTVISLRDTVALPENGASN ^(Me)GGAAAAQASSSCAQP v.061 tgg = N^(Me)AlaMSDITASRVESLDLQDLDLSELTVISLRDTVALPENGASN ^(Me) AGAAAAQASSSCAQP v.070tgg = W MSDITASRVESLDLQDLDLSELTVTSLRDTVALPENGASAWAAAAQASSSCAQP v.070tgg = N^(Me)Gly MSDITASRVESLDLQDLDLSELTVISLRDTVALPENGASAN ^(Me)GAAAAQASSSCAQP v.070 tgg = N^(Me)AlaMSDITASRVESLDLQDLDLSELTVISLRDTVALPENGASAN ^(Me) AAAAAQASSSCAQP v.086tgg = W MSDITASRVESLDLQDLDLSELTVTSLRDTVALPENGASAGWAAAQASSSCAQP v.086tgg = N^(Me)Gly MSDITASRVESLDLQDLDLSELTVISLRDTVALPENGASAGN ^(Me)GAAAQASSSCAQP v.086 tgg = N^(Me)AlaMSDITASRVESLDLQDLDLSELTVISLRDTVALPENGASAGN ^(Me) AAAAQASSSCAQP v.087tgg = W MSDITASRVESLDLQDLDLSELTVTSLRDTVALPENGASAGAWAAQASSSCAQP v.087tgg = N^(Me)Gly MSDITASRVESLDLQDLDLSELTVISLRDTVALPENGASAGAN ^(Me)GAAQASSSCAQP v.087 tgg = N^(Me)AlaMSDITASRVESLDLQDLDLSELTVISLRDTVALPENGASAGAN ^(Me) AAAQASSSCAQP v.088tgg = W MSDITASRVESLDLQDLDLSELTVTSLRDTVALPENGASAGAAWAQASSSCAQP v.088tgg = N^(Me)Ala MSDITASRVESLDLQDLDLSELTVISLRDTVALPENGASAGAAN ^(Me)AAQASSSCAQP v.089 tgg = WMSDITASRVESLDLQDLDLSELTVTSLRDTVALPENGASAGAAAWQASSSCAQP v.089tgg = N^(Me)Ala MSDITASRVESLDLQDLDLSELTVISLRDTVALPENGASAGAAAN ^(Me)AQASSSCAQP v.074 tgg = WMSDITASRVESLDLQDLDLSELTVISLRDTVALPENGASAGAAAAWASSSCAQP v.074tgg = N^(Me)Gly MSDITASRVESLDLQDLDLSELTVISLRDTVALPENGASAGAAAAN ^(Me)GASSSCAQP v.074 tgg = N^(Me)AlaMSDITASRVESLDLQDLDLSELTVISLRDTVALPENGASAGAAAAN ^(Me) AASSSCAQP v.074tgg = F₅Phe MSDITASRVESLDLQDLDLSELTVISLRDTVALPENGASAGAAAA_(f5) FASSSCAQPv.074 tgg = cLeu MSDITASRVESLDLQDLDLSELTVISLRDTVALPENGASAGAAAA_(c)LASSSCAQP v.074 tgg = ^(OH)AlaMSDITASRVESLDLQDLDLSELTVISLRDTVALPENGASAGAAAA^(OH) AASSSCAQP v.074tgg = ^(OH)Phe MSDITASRVESLDLQDLDLSELTVISLRDTVALPENGASAGAAAA^(OH)FASSSCAQP v.074 tgg = ^(D)AlaMSDITASRVESLDLQDLDLSELTVISLRDTVALPENGASAGAAAA^(D) AASSSCAQP v.075tgg = W MSDITASRVESLDLQDLDLSELTVTSLRDTVALPENGASAGAAAAAWSSSCAQP v.075tgg = N^(Me)Gly MSDITASRVESLDLQDLDLSELTVISLRDTVALPENGASAGAAAAAN ^(Me)GSSSCAQP v.075 tgg = N^(Me)AlaMSDITASRVESLDLQDLDLSELTVISLRDTVALPENGASAGAAAAAN ^(Me) ASSSCAQP v.077tgg = A MSDITASRVESLDLQDLDLSELTVTSLRDTVALPENGASAGAAAAQASSSCAQP v.077tgg = M MSDITASRVESLDLQDLDLSELTVISLRDTVALPENGASAGAAAAQASSSCMQPObservation results from LC-MS of thiopeptide derivative formed DNACodon Observation results from LC-MS of after Laz enzyme templatereprogramming precursor peptide after translation group treatmentRemarks v.061 tgg = W m/z = 1424.67 (z = 4) [Cys adduct]m/z = 682.28 (z = 2) ~45% Glu v.061 tgg = N^(Me)Glym/z = 1395.91 (z = 4) [Cys adduct] m/z = 624.72 (z = 2) misincorporationv.061 tgg = N^(Me)Ala m/z = 1399.41 (z = 4) [Cys adduct]m/z = 631.76 (z = 2) Partial hydrolysis v.070 tgg = Wm/z = 1428.17 (z = 4) [Cys adduct] m/z = 682.28 (z = 2) of precursorv.070 tgg = N^(Me)Gly m/z = 1399.40 (z = 4) [Cys adduct]m/z = 631.77 (z = 2) peptide v.070 tgg = N^(Me)Alam/z = 1402.90 (z = 4) [Cys adduct] m/z = 638.76 (z = 2)Partial hydrolysis v.086 tgg = W m/z = 1413.91 (z = 4) [βME adduct]m/z = 682.27 (z = 2) of precursor v.086 tgg = N^(Me)Glym/z = 1385.15 (z = 4) [βME adduct] m/z = 624.75 (z = 2) peptide v.086tgg = N^(Me)Ala m/z = 1388.66 (z = 4) [βME adduct] m/z = 631.75 (z = 2)v.087 tgg = W m/z = 1413.92 (z = 4) [βME adduct] m/z = 682.27 (z = 2)v.087 tgg = N^(Me)Gly m/z = 1385.16 (z = 4) [βME adduct]m/z = 624.74 (z = 2) v.087 tgg = N^(Me)Alam/z = 1388.67 (z = 4) [βME adduct] m/z = 631.75 (z = 2) v.088 tgg = Wm/z = 1413.92 (z = 4) [βME adduct] m/z = 682.27 (z = 2) v.088tgg = N^(Me)Ala m/z = 1388.66 (z = 4) [βME adduct] m/z = 631.75 (z = 2)v.089 tgg = W m/z = 1413.92 (z = 4) [βME adduct] m/z = 682.27 (z = 2)v.089 tgg = N^(Me)Ala m/z = 1388.66 (z = 4) [βME adduct]m/z = 631.75 (z = 2) v.074 tgg = W m/z = 1410.40 (z = 4) [Cys adduct]m/z = 653.74 (z = 2) v.074 tgg = N^(Me)Glym/z = 1381.65 (z = 4) [Cys adduct] m/z = 596.24 (z = 2) v.074tgg = N^(Me)Ala m/z = 1385.16 (z = 4) [Cys adduct] m/z = 603.24 (z = 2)v.074 tgg = F₅Phe m/z = 1423.14 (z = 4) [Cys adduct]m/z = 679.23 (z = 2) v.074 tgg = cLeu m/z = 1391.65 (z = 4) [Cys adduct]m/z = 616.23 (z = 2) v.074 tgg = ^(OH)Alam/z = 1381.91 (z = 4) [Cys adduct] m/z = 596.72 (z = 2) v.074tgg = ^(OH)Phe m/z = 1400.92 (z = 4) [Cys adduct] m/z = 634.73 (z = 2)v.074 tgg = ^(D)Ala m/z = 1381.66 (z = 4) [Cys adduct]m/z = 596.23 (z = 2) v.075 tgg = W m/z = 1410.42 (z = 4) [Cys adduct]m/z = 653.77 (z = 2) v.075 tgg = N^(Me)Glym/z = 1381.65 (z = 4) [Cys adduct] m/z = 596.25 (z = 2) v.075tgg = N^(Me)Ala m/z = 1385.15 (z = 4) [Cys adduct] m/z = 603.25 (z = 2)v.077 tgg = A m/z = 1366.15 (z = 4) m/z = 624.74 (z = 2) v.077 tgg = Mm/z = 1381.15 (z = 4) m/z = 654.74 (z = 2)

TABLE 43 DNA Codon template reprogrammingAmino acid sequence of precursor peptide v.080 tgg = WMSDITASRVESLDLQDLDLSELTVTSLRDTVALPENGASAGAAAAQASSSCAWP v.080tgg = N^(Me)Gly MSDITASRVESLDLQDLDLSELTVISLRDTVALPENGASAGAAAAQASSSCAN^(Me) GP v.081 tgg = WMSDITASRVESLDLQDLDLSELTVTSLRDTVALPENGASAGAAAAQASSSCAQW v.081tgg = N^(Me)Gly MSDITASRVESLDLQDLDLSELTVISLRDTVALPENGASAGAAAAQASSSCAQN^(Me) G v.081 tgg = N^(Me)AlaMSDITASRVESLDLQDLDLSELTVISLRDTVALPENGASAGAAAAQASSSCAQN ^(Me) A v.071tgg = W MSDITASRVESLDLQDLDLSELTVTSLRDTVALPENGASAWAAAWQASSSCAQP v.071tgg = N^(Me)Gly MSDITASRVESLDLQDLDLSELTVISLRDTVALPENGASAN ^(Me) GAAAN^(Me) GQASSSCAQP v.071 tgg = N^(Me)AlaMSDITASRVESLDLQDLDLSELTVISLRDTVALPENGASAN ^(Me) AAAAN ^(Me) AQASSSCAQPv.072 tgg = W MSDITASRVESLDLQDLDLSELTVTSLRDTVALPENGASAWAKAWQASSSCAQPaag = K v.072 tgg = N^(Me)Ala MSDITASRVESLDLQDLDLSELTVISLRDTVALPENGASAN^(Me) AAKAN ^(Me) AQASSSCAQP aag = K v.072 tgg = WMSDITASRVESLDLQDLDLSELTVISLRDTVALPENGASAWAN ^(Me) GAWQASSSCAQPaag = N^(Me)Gly v.072 tgg = N^(Me)AlaMSDITASRVESLDLQDLDLSELTVISLRDTVALPENGASAN ^(Me) AAN ^(Me) GAN ^(Me)AQASSSCAQP aag = N^(Me)Gly v.073 tgg = WMSDITASRVESLDLQDLDLSELTVTSLRDTVALPENGASWKWKWKQASSSCAQP aag = K v.073tgg = W MSDITASRVESLDLQDLDLSELTVISLRDTVALPENGASWN ^(Me) GWN ^(Me) GWN^(Me) GQASSSCAQP aag = N^(Me)Gly tgg = W v.100 cat = HMSDITASRVESLDLQDLDLSELTVISLRDTVALPENGASAWAHAFQASSSCAQN ^(Me) G ttt = Faag = N^(Me)Gly tgg = F₅Phe v.100 cat = cLeuMSDITASRVESLDLQDLDLSELTVISLRDTVALPENGASA_(f5) FA_(c) LAN ^(Me)AQASSSCAQN ^(Me) G ttt = N^(Me)Ala aag = N^(Me)Gly tgg = F₅Phe v.101cat = cLeu MSDITASRVESLDLQDLDLSELTVISLRDTVALPENGASAN ^(Me) GA_(f5) F_(c)CLQASSSCAQN ^(Me) A ttt = N^(Me)Ala aag = N^(Me)Gly v.064 N. A.MSDITASRVESLDLQDLDLSELTVTSLRDTVALPENGASWGAAAAAAQASSSCAQPNNLNVG v.082N. A. MSDITASRVESLDLQDLDLSELTVTSLRDTVALPENGASAGAAAAQASSSCAQPGELARPObservation results from LC-MS of Observation resultsthiopeptide derivative DNA Codon from LC-MS of precursorformed after Laz template reprogramming peptide after translationenzyme group treatment Remarks v.080 tgg = W m/z = 1380.68 (z = 4)m/z = 653.75 (z = 2) HPLC tailing v.080 tgg = N^(Me)Glym/z = 1351.91 (z = 4) m/z = 596.24 (z = 2) of thiopeptide v.081 tgg = Wm/z = 1388.41 (z = 4) m/z = 669.27 (z = 2) v.081 tgg = N^(Me)Glym/z = 1359.66 (z = 4) m/z = 611.74 (z = 2) v.081 tgg = N^(Me)Alam/z = 1363.16 (z = 4) m/z = 618.75 (z = 2) v.071 tgg = Wm/z = 1456.92 (z = 4) [Cys adduct] m/z = 746.80 (z = 2) v.071tgg = N^(Me)Gly m/z = 1399.40 (z = 4) [Cys adduct] m/z = 631.76 (z = 2)v.071 tgg = N^(Me)Ala m/z = 1406.42 (z = 4) [Cys adduct]m/z = 645.77 (z = 2) v.072 tgg = W m/z = 1471.18 (z = 4) [Cys adduct]m/z = 775.32 (z = 2) aag = K v.072 tgg = N^(Me)Alam/z = 1420.69 (z = 4) [Cys adduct] m/z = 674.31 (z = 2) aag = K v.072tgg = W m/z = 1456.94 (z = 4) [Cys adduct] m/z = 746.80 (z = 2)aag = N^(Me)Gly v.072 tgg = N^(Me)Ala m/z = 1406.43 (z = 4) [Cys adduct]m/z = 645.78 (z = 2) aag = N^(Me)Gly v.073 tgg = W N. D.m/z = 593.59 (z = 3) aag = K v.073 tgg = Wm/z = 1485.68 (z = 4) [Cys adduct] m/z = 804.32 (z = 2) aag = N^(Me)Glytgg = W v.100 cat = H m/z = 1457.18 (z = 4) [Cys adduct]m/z = 747.30 (z = 2) ttt = F aag = N^(Me)Gly tgg = F₅Phe v.100cat = cLeu m/z = 1447.92 (z = 4) [Cys adduct] m/z = 728.77 (z = 2)ttt = N^(Me)Ala aag = N^(Me)Gly tgg = F₅Phe v.101 cat = cLeum/z = 1447.93 (z = 4) [Cys adduct] m/z = 728.75 (z = 2) ttt = N^(Me)Alaaag = N^(Me)Gly v.064 N. A. m/z = 1613.02 (z = 4) [Cys adduct]m/z = 1058.95 (z = 2) v.082 N. A. m/z = 1541.00 (z = 4) [βME adduct]m/z = 936.42 (z = 2)

TABLE 44 DNA Codon template reprogrammingAmino acid sequence of precursor peptide v.088 tgg = N^(Me)GlyMSDITASRVESLDLQDLDLSELTVTSLRDTVALPENGASAGAAN ^(Me) GAQASSSCAQP v.089tgg = N^(M)eGly MSDITASRVESLDLQDLDLSELTVTSLRDTVALPENGASAGAAAN ^(Me)GQASSSCAQP v.074 tgg = ^(D)SerMSDITASRVESLDLQDLDLSELTVTSLRDTVALPENGASAGAAAA^(D) SASSSCAQP v.077tgg = F MSDITASRVESLDLQDLDLSELTVTSLRDTVALPENGASAGAAAAQASSSCFQP v.077tgg = R MSDITASRVESLDLQDLDLSELTVTSLRDTVALPENGASAGAAAAQASSSCRQP v.080tgg = N^(Me)Ala MSDITASRVESLDLQDLDLSELTVTSLRDTVALPENGASAGAAAAQASSSCAN^(Me) AP tgg = W v.100 cat = HMSDITASRVESLDLQDLDLSELTVTSLRDTVALPENGASAWAHAFQASSSCAQK ttt = F aag = Ktgg = W v.100 cat = cLeuMSDITASRVESLDLQDLDLSELTVTSLRDTVALPENGASAWAcLAFQASSSCAQK ttt = F aag = KObservation results from LC-MS of Observation results fromthiopeptide derivative DNA Codon LC-MS of precursor formed after Laztemplate reprogramming peptide after translation enzyme group treatmentRemarks v.088 tgg = N^(Me)Gly m/z = 1385.16 (z = 4) [βmE adduct]m/z = 624.75 (z = 2) v.089 tgg = N^(M)eGlym/z = 1385.17 (z = 4) [βmE adduct] m/z = 624.75 (z = 2) v.074tgg = ^(D)Ser m/z = 1385.66 (z = 4) [Cys adduct] m/z = 604.22 (z = 2)v.077 tgg = F m/z = 1385.17 (z = 4) m/z = 662.77 (z = 2) v.077 tgg = Rm/z = 1387.42 (z = 4) m/z = 667.29 (z = 2) v.080 tgg = N^(Me)Alam/z = 1355.42 (z = 4) m/z = 603.24 (z = 2) tgg = W v.100 cat = Hm/z = 1177.34 (z = 5) [Cys adduct] m/z = 775.83 (z = 2) ttt = F aag = Ktgg = W v.100 cat = cLeu m/z = 1464.95 (z = 4) [Cys adduct]m/z = 762.84 (z = 2) ttt = F aag = K

[Example 14-2] Substrate Tolerance of Macrocyclization toNon-Proteinogenic Amino Acid-Substituted Analog N^(Me)Gly

As in Example 9-2, W2N^(Me)Gly, G3N^(Me)Gly, A4N^(Me)Gly, A5N^(Me)Gly,A6N^(Me)Gly, A7N^(Me)Gly, Q8N^(Me)Gly, A9N^(Me)Gly, C13N^(Me)Gly,A14N^(Me)Gly, Q15N^(Me)Gly and P16N^(Me)Gly altered precursor peptides(see FIG. 50 ) were synthesized by replacing the 2nd to 9th and 13th to16th native amino acids, respectively, with N^(Me)Gly. For convenienceof reprogramming of genetic codes, the N-terminal residues of theprecursor peptides were changed to biotinylated phenylalanine (^(bio)F),not formylmethionine (fMet). Subsequently, each altered precursorpeptide mentioned above was reacted in accordance with the proceduresdescribed in Example 9-2, and the reaction product was analyzed byLC-MS. Modification reaction mediated by the Laz enzyme group and theformation of macrocyclization products were confirmed by broad extractedion chromatograms (brEIC) in which peaks detected in the range of ±400of the tetravalent m/z value of a precursor peptide were integrated.FIG. 50 shows brEIC of each reaction product.

[Example 14-3] Substrate Tolerance of Macrocyclization toNon-Proteinogenic Amino Acid-Substituted Analog N^(Me)Ala

As in Example 9-2, W2N^(Me)Ala, G3N^(Me)Ala, A4N^(Me)Ala, A5N^(Me)Ala,A6N^(Me)Ala, A7N^(Me)Ala, Q8N^(Me)Ala, A9N^(Me)Ala, C13N^(Me)Ala,A14N^(Me)Ala, Q15N^(Me)Ala and P16N^(Me)Ala altered precursor peptides(see FIG. 51 ) were synthesized by replacing the 2nd to 9th and 13th to16th native amino acids, respectively, with N^(Me)Ala. For convenienceof reprogramming of genetic codes, the N-terminal residues of theprecursor peptides were changed to biotinylated phenylalanine (^(bio)F),not formylmethionine (fMet). Subsequently, each altered precursorpeptide mentioned above was reacted in accordance with the proceduresdescribed in Example 9-2, and the reaction product was analyzed byLC-MS. Modification reaction mediated by the Laz enzyme group and theformation of macrocyclization products were confirmed by broad extractedion chromatograms (brEIC) in which peaks detected in the range of ±400of the tetravalent m/z value of a precursor peptide were integrated.FIG. 51 shows brEIC of each reaction product.

[Example 15] Reaction of Precursor Peptide Displayed on mRNA

In order to reveal that a precursor peptide displayed on mRNA would bemodified by lactazole biosynthetic enzymes, the experiment outlined inFIG. 52 was conducted. A DNA linker labeled with puromycin was ligated,using T4 RNA ligase, to a 3′ region of a mRNA encoding a precursorpeptide. A mixture of the precursor peptide and the precursor peptidedisplayed on the mRNA was obtained by translational synthesis with thismRNA labeled with puromycin used as a template. From this mixture, theprecursor peptide displayed on mRNA was isolated by using biotinylatedDNA complementary to the mRNA, and streptavidin beads. Modificationreaction was carried out by its incubation with the Laz enzyme group,and the products were analyzed by LC-MS. As a result, as shown in FIG.53 , leader peptide fragments formed when the modification reactionproceeded was able to be detected, demonstrating that the precursorpeptide displayed on mRNA is modified by the lactazole biosyntheticenzymes.

[Example 16] Study of Influence of Order of Addition of Enzymes onReaction

By changing the order of addition of enzymes to a reaction system,influence of it on the reaction of a precursor peptide was studied.

The reaction of precursor peptide LazA* in a cell-free translationsystem was attempted according to the enzyme addition patterns I, II,III and IV shown in FIG. 54 . The pattern I involved first adding LazD,LazE and LazF, followed by reaction at 25° C. for 16 hours, next addingLazB, cosubstrate tRNA^(Glu) and aminoacylation enzyme GluRS, followedby reaction at 25° C. for 6 hours, and then adding LazC, followed byreaction at 25° C. for 0.5 hours. The pattern II involved first addingLazD, LazE, LazF, LazB, cosubstrate tRNA^(Glu) and aminoacylation enzymeGluRS, followed by reaction at 25° C. for 16 hours, and then addingLazC, followed by reaction at 25° C. for 0.5 hours. The pattern IIIinvolved first adding LazD, LazE, and LazF, followed by reaction at 25°C. for 6 hours, and then adding LazB, cosubstrate tRNA^(Glu),aminoacylation enzyme GluRS and LazC, followed by reaction at 25° C. for12 hours. The pattern IV involved adding LazD, LazE, LazF, LazB,cosubstrate tRNA^(Glu), aminoacylation enzyme GluRS and LazC, followedby reaction at 25° C. for 16 hours.

The results about I, II, III and IV are shown in FIG. 54

1. A method for producing a compound library comprising two or morecyclic compounds represented by the formula (I):

wherein m number of X_(a), and X_(b) and X_(c) each independentlyrepresent an amino acid residue selected from the group consisting ofany amino acids and analogs thereof, Z is present or absent and, whenpresent, represents a peptide consisting of 1 to 100 amino acids and/oranalogs thereof, m is an integer selected from 2 to 40, ring A is anitrogen-containing 6-membered ring optionally having a substituent, B¹is an oxygen atom, a sulfur atom, or a NH group, and R¹ is a hydrogenatom or a hydrocarbon group, the method comprising a step of allowing amacrocyclase in vitro to act on two or more peptides represented by theformula (II): $\begin{matrix}{{LP} - X - ( {Xa} )_{m} - Y - Z} & ({II})\end{matrix}$ wherein X represents a group represented by the formula(1):

wherein R² is a hydrogen atom or a hydrocarbon group, Y is a peptideresidue consisting of four amino acids and/or analogs thereof andcontains a group represented by the formula (2):

wherein R¹ and B¹ are as defined above, and R³ represents a hydrogen ora hydrocarbon group, m number of X_(a), m and Z are as defined above,and LP is present or absent and, when present, represents a peptideresidue consisting of 1 to 100 amino acids and/or analogs thereof, andforming the nitrogen-containing 6-membered ring A while eliminating LP,if present, to form the two or more cyclic compounds represented by theformula (I).
 2. The production method according to claim 1, wherein LPis a peptide residue consisting of 11 or more and 100 or less aminoacids and/or analogs thereof.
 3. The production method according toclaim 1, wherein m is an integer selected from 2 to
 24. 4. Theproduction method according to claim 1, wherein Y is a group representedby the following formula (3):

wherein R¹, R³, R⁴ and R⁵ each independently represent a hydrogen atomor a hydrocarbon group, and B¹ and B² each independently represent anoxygen atom, a sulfur atom or a NH group.
 5. The production methodaccording to claim 1, wherein Y is a group represented by the followingformula (3′):

wherein R¹, R³, R⁴ and R⁵ each independently represent a hydrogen atomor a methyl group, and B¹ and B² each independently represent an oxygenatom or a sulfur atom, with the proviso that when B¹ and/or B² is asulfur atom, each of R¹ and R⁵ is a hydrogen.
 6. The production methodaccording to claim 1, wherein Y is a group represented by the followingformula (3″):

wherein R¹, R⁴ and R⁵ each independently represent a hydrogen atom or amethyl group, and B¹ and B² each independently represent an oxygen atomor a sulfur atom, with the proviso that when B¹ and/or B² is a sulfuratom, each of R¹ and R⁵ is a hydrogen.
 7. The production methodaccording to claim 1, wherein Y is a group represented by the followingformula (3-1):


8. The production method according to claim 1, wherein the macrocyclasecomprises LazC and/or an enzyme having homology with LazC.
 9. A methodfor producing a compound library comprising two or more cyclic compoundsrepresented by the formula (I):

wherein m number of X_(a), and X_(b) and X_(c) each independentlyrepresent an amino acid residue selected from the group consisting ofany amino acids and analogs thereof, Z is present or absent and, whenpresent, represents a peptide consisting of 1 to 100 amino acids and/oranalogs thereof, m is an integer selected from 2 to 40, ring A is anitrogen-containing 6-membered ring optionally having a substituent, B¹is an oxygen atom, a sulfur atom, or a NH group, and R¹ is a hydrogenatom or a hydrocarbon group, the method comprising: a step of producinga mRNA library encoding precursor peptides represented by the formula(III): $\begin{matrix}{{LP}^{\prime} - X^{\prime} - ( {Xa}^{\prime} )_{m^{\prime}} - Y^{\prime} - Z^{\prime}} & ({III})\end{matrix}$ wherein X′ is serine or threonine, or an analog thereof,Y′ is a peptide consisting of four amino acids represented by—Y′(10)-Y′(11)-Y′(12)-Y′(13)- and/or analogs thereof, wherein Y′(10) isan amino acid residue selected from the group consisting of any aminoacids and analogs thereof, Y′(11) is serine, cysteine, threonine ordiaminopropionic acid, or an analog thereof, Y′(12) is serine orthreonine, or an analog thereof, and Y′(13) is an amino acid residueselected from the group consisting of any amino acids and analogsthereof, m′ number of Xa′, m′ and Z′ are the same as defined as m numberof Xa, m and Z, respectively, in the formula (I), and LP′ is present orabsent and, when present, represents a peptide consisting of 1 to 100amino acids and/or analogs thereof; a step of expressing the precursorpeptides by a cell-free translation system with the mRNA library toproduce a first peptide library; a step of reacting an azolering-forming enzyme with the first peptide library, so that an azolering is formed on at least Y′(11) in the precursor peptides to produce asecond peptide library; a step of reacting an α,β-unsaturated aminoacid-forming enzyme with the second peptide library in the presence ofcosubstrate tRNA^(Glu) for glutamylation reaction and aminoacylationenzyme GluRS, so that at least X′ and Y′(12) in the precursor peptidesare converted to α,β-unsaturated amino acid residues to produce a thirdpeptide library; and a step of reacting a macrocyclase with the thirdpeptide library and forming a nitrogen-containing 6-membered ring whileeliminating LP′, if present, to form the two or more cyclic compoundsrepresented by the formula (I).
 10. A method for producing a compoundlibrary comprising two or more cyclic compounds represented by theformula (I):

wherein m number of X_(a), and X_(b) and X_(c) each independentlyrepresent an amino acid residue selected from the group consisting ofany amino acids and analogs thereof, Z is present or absent and, whenpresent, represents a peptide consisting of 1 to 100 amino acids and/oranalogs thereof, m is an integer selected from 2 to 40, ring A is anitrogen-containing 6-membered ring optionally having a substituent, B¹is an oxygen atom, a sulfur atom, or a NH group, and R¹ is a hydrogenatom or a hydrocarbon group, the method comprising: a step of producinga mRNA library encoding precursor peptides represented by the formula(III): $\begin{matrix}{{LP}^{\prime} - X^{\prime} - ( {Xa}^{\prime} )_{m^{\prime}} - Y^{\prime} - Z^{\prime}} & ({III})\end{matrix}$ wherein X′ is serine or threonine, or an analog thereof,Y′ is a peptide consisting of four amino acids represented by—Y′(10)-Y′(11)-Y′(12)-Y′(13)- and/or analogs thereof, wherein Y′(10) isan amino acid residue selected from the group consisting of any aminoacids and analogs thereof, Y′(11) is serine, cysteine, threonine ordiaminopropionic acid, or an analog thereof, Y′(12) is serine orthreonine, or an analog thereof, and Y′(13) is an amino acid residueselected from the group consisting of any amino acids and analogsthereof, m′ number of Xa′, m′ and Z′ are the same as defined as m numberof Xa, m and Z, respectively, in the formula (I), and LP′ is present orabsent and, when present, represents a peptide consisting of 1 to 100amino acids and/or analogs thereof; a step of binding puromycin to the3′ end of each mRNA of the mRNA library to produce a puromycin-boundmRNA library; a step of expressing the precursor peptides by a cell-freetranslation system with the puromycin-bound mRNA library to produce afirst peptide-mRNA complex library; a step of reacting an azolering-forming enzyme with the first peptide-mRNA complex library, so thatan azole ring is formed on at least Y′(11) in the precursor peptides toproduce a second peptide-mRNA complex library; a step of reacting anα,β-unsaturated amino acid-forming enzyme with the second peptide-mRNAcomplex library in the presence of cosubstrate tRNA^(Glu) forglutamylation reaction and aminoacylation enzyme GluRS, so that at leastX′ and Y′(12) in the precursor peptides are converted to α,β-unsaturatedamino acid residues to produce a third peptide-mRNA complex library; anda step of reacting a macrocyclase with the third peptide-mRNA complexlibrary and forming a nitrogen-containing 6-membered ring whileeliminating LP′, if present, to form the two or more cyclic compoundsrepresented by the formula (I). 11-12. (canceled)
 13. The productionmethod according to claim 9, wherein Y′(10) is serine or threonine, oran analog thereof, and Y′(13) is serine, cysteine, threonine ordiaminopropionic acid, or an analog thereof.
 14. The production methodaccording to claim 9, wherein Y′(11) is serine, cysteine or threonine,or an analog thereof, and Y′(12) is serine or threonine, or an analogthereof.
 15. (canceled)
 16. The production method according to claim 9,wherein Y′(10) is serine or threonine, or an analog thereof, Y′(11) isserine, cysteine or threonine, or an analog thereof, Y′(12) is serine,and Y′(13) is serine, cysteine or threonine, or an analog thereof. 17.The production method according to claim 9, wherein in (Xa′)_(m′) in theformula (III), amino acid residue Xa′(1) adjacent to X′ is an amino acidother than an acidic amino acid or an analog thereof.
 18. The productionmethod according to claim 9, wherein in (Xa′)_(m′) in the formula (III),amino acid residue Xa′(m′) at m′-th position is an amino acid other thanan acidic amino acid or an analog thereof.
 19. The production methodaccording to claim 9, wherein in (Xa′)_(m′) in the formula (III), aminoacid residue Xa′(m′−1) at (m′−1)-th position is an amino acid other thanan acidic amino acid and a basic amino acid or an analog thereof. 20.The production method according to claim 9, wherein the azolering-forming enzyme comprises LazD, LazE and LazF, and/or an enzymehaving homology with any of them.
 21. The production method according toclaim 9, wherein the α,β-unsaturated amino acid-forming enzyme comprisesLazB and LazF, and/or an enzyme having homology with any of them. 22.The production method according to claim 9, wherein the macrocyclasecomprises LazC and/or an enzyme having homology with LazC.
 23. Theproduction method according to claim 9, wherein the cosubstratetRNA^(Glu) for glutamylation reaction is actinomycete-derivedtRNA^(Glu), and the aminoacylation enzyme GluRS is actinomycete-derivedGluRS. 24-28. (canceled)